Ultra bright dimeric or polymeric dyes with spacing linker groups

ABSTRACT

or a stereoisomer, tautomer or salt thereof, wherein R1, R2, R3, R4, R5, L1, L2, L3, L4, M, m and n are as defined herein. Methods associated with preparation and use of such compounds are also provided.

BACKGROUND Field

The present invention is generally directed to dimeric and polymericfluorescent or colored dyes having rigid spacing groups, and methods fortheir preparation and use in various analytical methods.

Description of the Related Art

Fluorescent and/or colored dyes are known to be particularly suitablefor applications in which a highly sensitive detection reagent isdesirable. Dyes that are able to preferentially label a specificingredient or component in a sample enable the researcher to determinethe presence, quantity and/or location of that specific ingredient orcomponent. In addition, specific systems can be monitored with respectto their spatial and temporal distribution in diverse environments.

Fluorescence and colorimetric methods are extremely widespread inchemistry and biology. These methods give useful information on thepresence, structure, distance, orientation, complexation and/or locationfor biomolecules. In addition, time-resolved methods are increasinglyused in measurements of dynamics and kinetics. As a result, manystrategies for fluorescence or color labeling of biomolecules, such asnucleic acids and protein, have been developed. Since analysis ofbiomolecules typically occurs in an aqueous environment, the focus hasbeen on development and use of water soluble dyes.

Highly fluorescent or colored dyes are desirable since use of such dyesincreases the signal to noise ratio and provides other related benefits.Accordingly, attempts have been made to increase the signal from knownfluorescent and/or colored moieties. For example, dimeric and polymericcompounds comprising two or more fluorescent and/or colored moietieshave been prepared in anticipation that such compounds would result inbrighter dyes. However, as a result of intramolecular fluorescencequenching, the known dimeric and polymeric dyes have not achieved thedesired increase in brightness.

There is thus a need in the art for water soluble dyes having anincreased molar brightness. Ideally, such dyes and biomarkers should beintensely colored or fluorescent and should be available in a variety ofcolors and fluorescent wavelengths. The present invention fulfills thisneed and provides further related advantages.

BRIEF SUMMARY

In brief, embodiments of the present invention are generally directed tocompounds useful as water soluble, fluorescent and/or colored dyesand/or probes that enable visual detection of analyte molecules, such asbiomolecules, as well as reagents for their preparation. Methods forvisually detecting analyte molecules using the dyes are also described.

Embodiments of the presently disclosed dyes include two or morefluorescent and/or colored moieties covalently linked by a linker(“L⁴”). In contrast to previous reports of dimeric and/or polymericdyes, the present dyes are significantly brighter than the correspondingmonomeric dye compound. While, not wishing to be bound by theory, it isbelieved that the linker moiety provides sufficient spatial separationbetween the fluorescent and/or colored moieties such that intramolecularfluorescence quenching is reduced and/or eliminated.

The water soluble, fluorescent or colored dyes of embodiments of theinvention are intensely colored and/or fluorescent and can be readilyobserved by visual inspection or other means. In some embodiments thecompounds may be observed without prior illumination or chemical orenzymatic activation. By appropriate selection of the dye, as describedherein, visually detectable analyte molecules of a variety of colors maybe obtained.

In one embodiment, compounds having the following structure (I) areprovided:

or a stereoisomer, tautomer or salt thereof, wherein R¹, R², R³, R⁴, R⁵,L¹, L², L³, L⁴, M, m and n are as defined herein. Compounds of structure(I) find utility in a number of applications, including use asfluorescent and/or colored dyes in various analytical methods.

In another embodiment, a method for staining a sample is provided, themethod comprises adding to said sample a compound of structure (I) in anamount sufficient to produce an optical response when said sample isilluminated at an appropriate wavelength.

In still other embodiments, the present disclosure provides a method forvisually detecting an analyte molecule, comprising:

(a) providing a compound of (I); and

(b) detecting the compound by its visible properties.

Other disclosed methods include a method for visually detecting abiomolecule, the method comprising:

(a) admixing a compound of structure (I) with one or more biomolecules;and

(b) detecting the compound by its visible properties.

Other embodiments provide a method for visually detecting an analyte,the method comprising:

-   -   (a) providing a compound as disclosed herein, wherein R² or R³        comprises a linker comprising a covalent bond to a targeting        moiety having specificity for the analyte;    -   (b) admixing the compound and the analyte, thereby associating        the targeting moiety and the analyte; and    -   (c) detecting the compound by its visible properties.

Other embodiments are directed to a composition comprising a compound ofstructure (I) and one or more analyte molecule, such as a biomolecule.Use of such compositions in analytical methods for detection of the oneor more biomolecules is also provided.

In some other different embodiments is provided a compound of structure(II):

or a stereoisomer, salt or tautomer thereof, wherein R¹, R², R³, R⁴, R⁵,L^(1a), L², L³, L⁴, G, m and n are as defined herein. Compounds ofstructure (II) find utility in a number of applications, including useas intermediates for preparation of fluorescent and/or colored dyes ofstructure (I).

In yet other embodiments a method for labeling an analyte molecule isprovided, the method comprising:

(a) admixing a compound of structure (II), wherein R² or R³ is Q or alinker comprising a covalent bond to Q, with the analyte molecule;

(b) forming a conjugate of the compound and the analyte molecule; and

(c) reacting the conjugate with a compound of formula M-L^(1b)-G′,thereby forming at least one covalent bond by reaction of G and G′,wherein R², R³, Q, G and M-L^(1b)-G′ are as defined herein.

In some different embodiments another method for labeling an analytemolecule is provided, the method comprising:

(a) admixing a compound of structure (II), wherein R² or R³ is Q or alinker comprising a covalent bond to Q, with a compound of formulaM-L^(1b)-G′, thereby forming at least one covalent bond by reaction of Gand G′; and

(b) reacting the product of step (A) with the analyte molecule, therebyforming a conjugate of the product of step (A) and the analyte moleculewherein R², R³, Q, G and M-L^(1b)-G′ are as defined herein.

In more different embodiments, a method for preparing a compound ofstructure (I) is provided, the method comprising admixing a compound ofstructure (II) with a compound of formula M-L^(1b)-G′, thereby formingat least one covalent bond by reaction of G and G′, wherein G andM-L^(1b)-G′ are as defined herein.

Still more embodiments are directed to a fluorescent compound comprisingY fluorescent moieties M, wherein the fluorescent compound has a peakfluorescence emission upon excitation with a predetermined wavelength ofultraviolet light of at least 85% of Y times greater than the peakfluorescence emission of a single M moiety upon excitation with the samewavelength of ultraviolet light, and wherein Y is an integer of 2 ormore.

These and other aspects of the invention will be apparent upon referenceto the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

In the figures, identical reference numbers identify similar elements.The sizes and relative positions of elements in the figures are notnecessarily drawn to scale and some of these elements are arbitrarilyenlarged and positioned to improve figure legibility. Further, theparticular shapes of the elements as drawn are not intended to conveyany information regarding the actual shape of the particular elements,and have been solely selected for ease of recognition in the figures.

FIG. 1 provides UV absorbance spectra for representative compoundscomprising a triethylene glycol spacer and a comparative compound at 5μm and pH 9.

FIG. 2 is UV absorbance data for representative compounds comprising ahexaethylene glycol spacer and a comparative compound at 5 μm and pH 9.

FIG. 3 is fluorescence emission spectra for representative compoundscomprising a triethylene glycol spacer and a comparative compound at 50nM and pH 9.

FIG. 4 presents fluorescence emission spectra for representativecompounds comprising a hexaethylene glycol spacer and a comparativecompound at 50 nM and pH 9.

FIG. 5 is UV absorbance data at 5 μm for representative compoundscomprising four hexaethylene glycol spacers and two or three fluoresceinmoieties relative to a comparative compound having a single fluoresceinmoiety.

FIG. 6 is a graph of fluorescent emission data at 5 μm forrepresentative compounds comprising four hexaethylene glycol spacers andtwo or three fluorescein moieties relative to a comparative compoundhaving a single fluorescein moiety.

FIG. 7 shows comparative fluorescence emission response for illustrativecompounds with various m values.

FIG. 8 provides data comparing fluorescence emission for the “HEG”compound, wherein m is 1, 2 or 3, relative to Compound A.

FIG. 9 provides UV absorbance data for compound I-32, compound I-46 andCompound B.

FIG. 10 shows the results of a reaction trimerizing compound I-42 asanalyzed by PAGE.

FIG. 11 provides data comparing the fluorescence signal of sevencompounds in a dead and necrotic cell population.

FIG. 12 shows fluorescence intensity of an antibody conjugate of I-51versus an antibody conjugate of Compound G.

FIG. 13 shows comparisons of an I-51 conjugation and a Compound Greference antibody.

FIG. 14 shows a comparison of UCHT1-I-51, UCHT1-BB515, and UCHT1-FITC.

FIG. 15 shows expression levels of CD3 compared to a MEF standard curve.

FIG. 16 shows a comparison of UCHT1-I-16 fractions to FITC.

FIG. 17 shows a comparison of UCHT1-I-16 fractions to I-56 conjugates.

FIG. 18 shows a comparison of the UCHT1-I-51-like analogue, UCHT1 I-16,with UCHT1 I-56 (10×), and UCHT1 I-53 (6×).

FIG. 19 provides data comparing the UCHT1 I-51-like analogue, UCHT1I-16, was compared with UCHT1 I-56 (10×), and UCHT1 I-53 (6×).

FIG. 20 shows the results of a regression analysis performed on dataproduced when testing UCHT1 I-16 and UCHT1 I-49 conjugates todemonstrate equivalency between conjugations.

FIG. 21A shows correlations between I-16 and I-45 as determined usingregression analysis. FIG. 21B shows titration curve overlays andcompared to references.

FIG. 21C shows example qualitative data showing background FL and cellmorphology comparing Compound D and I-45.

FIG. 22 shows affinity curves, as histograms, with compound emissiondetected in the FL1-A channel.

FIG. 23A shows comparisons of fluorescence intensity of off target,non-specific binding of UCHT1-I-21B, UCHT1-I-16, and reference,UCHT1-FITC, and FIG. 23B presents supporting data.

FIG. 24 presents results of a regression analysis that was applied tothe data to review correlations and relative affinities.

FIG. 25, shows signal to noise data for UCHT1-I-21B, UCHT1-I-51, andUCHT1-FITC.

FIGS. 26A and 26B provide data comparing UCHT1 Compound G AND UCHT1 I-51in a plasma interference study using PBMC. FIG. 26A shows data resultingfrom the addition of 0% glycine, and FIG. 26B shows data resulting fromthe addition of 2.5% glycine.

DETAILED DESCRIPTION

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments of theinvention. However, one skilled in the art will understand that theinvention may be practiced without these details.

Unless the context requires otherwise, throughout the presentspecification and claims, the word “comprise” and variations thereof,such as, “comprises” and “comprising” are to be construed in an open,inclusive sense, that is, as “including, but not limited to”.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

“Amino” refers to the —NH₂ group.

“Carboxy” refers to the —CO₂H group.

“Cyano” refers to the —CN group.

“Formyl” refers to the —C(═O)H group.

“Hydroxy” or “hydroxyl” refers to the —OH group.

“Imino” refers to the ═NH group.

“Nitro” refers to the —NO₂ group.

“Oxo” refers to the ═O substituent group.

“Sulfhydryl” refers to the —SH group.

“Thioxo” refers to the ═S group.

“Alkyl” refers to a straight or branched hydrocarbon chain groupconsisting solely of carbon and hydrogen atoms, containing nounsaturation, having from one to twelve carbon atoms (C₁-C₁₂ alkyl), oneto eight carbon atoms (C₁-C₈ alkyl) or one to six carbon atoms (C₁-C₆alkyl), and which is attached to the rest of the molecule by a singlebond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl),n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl,2-methylhexyl, and the like. Unless stated otherwise specifically in thespecification, alkyl groups are optionally substituted.

“Alkylene” or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain linking the rest of the molecule to a radical group,consisting solely of carbon and hydrogen, containing no unsaturation,and having from one to twelve carbon atoms, e.g., methylene, ethylene,propylene, n-butylene, ethenylene, propenylene, n-butenylene,propynylene, n-butynylene, and the like. The alkylene chain is attachedto the rest of the molecule through a single bond and to the radicalgroup through a single bond. The points of attachment of the alkylenechain to the rest of the molecule and to the radical group can bethrough one carbon or any two carbons within the chain. Unless statedotherwise specifically in the specification, alkylene is optionallysubstituted.

“Alkenylene” or “alkenylene chain” refers to a straight or brancheddivalent hydrocarbon chain linking the rest of the molecule to a radicalgroup, consisting solely of carbon and hydrogen, containing at least onecarbon-carbon double bond and having from two to twelve carbon atoms,e.g., ethenylene, propenylene, n-butenylene, and the like. Thealkenylene chain is attached to the rest of the molecule through asingle bond and to the radical group through a double bond or a singlebond. The points of attachment of the alkenylene chain to the rest ofthe molecule and to the radical group can be through one carbon or anytwo carbons within the chain. Unless stated otherwise specifically inthe specification, alkenylene is optionally substituted.

“Alkynylene” or “alkynylene chain” refers to a straight or brancheddivalent hydrocarbon chain linking the rest of the molecule to a radicalgroup, consisting solely of carbon and hydrogen, containing at least onecarbon-carbon triple bond and having from two to twelve carbon atoms,e.g., ethenylene, propenylene, n-butenylene, and the like. Thealkynylene chain is attached to the rest of the molecule through asingle bond and to the radical group through a double bond or a singlebond. The points of attachment of the alkynylene chain to the rest ofthe molecule and to the radical group can be through one carbon or anytwo carbons within the chain. Unless stated otherwise specifically inthe specification, alkynylene is optionally substituted.

“Alkylether” refers to any alkyl group as defined above, wherein atleast one carbon-carbon bond is replaced with a carbon-oxygen bond. Thecarbon-oxygen bond may be on the terminal end (as in an alkoxy group) orthe carbon oxygen bond may be internal (i.e., C—O—C). Alkylethersinclude at least one carbon oxygen bond, but may include more than one.For example, polyethylene glycol (PEG) is included within the meaning ofalkylether. Unless stated otherwise specifically in the specification,an alkylether group is optionally substituted. For example, in someembodiments an alkylether is substituted with an alcohol or—OP(═R_(a))(R_(b))R_(c), wherein each of R_(a), R_(b) and R_(c) is asdefined for compounds of structure (I).

“Alkoxy” refers to a group of the formula —OR_(a) where R_(a) is analkyl group as defined above containing one to twelve carbon atoms.Unless stated otherwise specifically in the specification, an alkoxygroup is optionally substituted.

“Alkoxyalkylether” refers to a group of the formula —OR_(a)R_(b) whereR_(a) is an alkylene group as defined above containing one to twelvecarbon atoms, and R_(b) is an alkylether group as defined herein. Unlessstated otherwise specifically in the specification, an alkoxyalkylethergroup is optionally substituted, for example substituted with an alcoholor —OP(═R_(a))(R_(b))R_(c), wherein each of R_(a), R_(b) and R_(c) is asdefined for compounds of structure (I).

“Heteroalkyl” refers to an alkyl group, as defined above, comprising atleast one heteroatom (e.g., N, O, P or S) within the alkyl group or at aterminus of the alkyl group. In some embodiments, the heteroatom iswithin the alkyl group (i.e., the heteroalkyl comprises at least onecarbon-[heteroatom]_(x)-carbon bond, where x is 1, 2 or 3). In otherembodiments, the heteroatom is at a terminus of the alkyl group and thusserves to join the alkyl group to the remainder of the molecule (e.g.,M1-H-A), where M1 is a portion of the molecule, H is a heteroatom and Ais an alkyl group). Unless stated otherwise specifically in thespecification, a heteroalkyl group is optionally substituted. Exemplaryheteroalkyl groups include ethylene oxide (e.g., polyethylene oxide),optionally including phosphorous-oxygen bonds, such as phosphodiesterbonds.

“Heteroalkoxy” refers to a group of the formula —OR_(a) where R_(a) is aheteroalkyl group as defined above containing one to twelve carbonatoms. Unless stated otherwise specifically in the specification, aheteroalkoxy group is optionally substituted.

“Heteroalkylene” refers to an alkylene group, as defined above,comprising at least one heteroatom (e.g., N, O, P or S) within thealkylene chain or at a terminus of the alkylene chain. In someembodiments, the heteroatom is within the alkylene chain (i.e., theheteroalkylene comprises at least one carbon-[heteroatom]-carbon bond,where x is 1, 2 or 3). In other embodiments, the heteroatom is at aterminus of the alkylene and thus serves to join the alkylene to theremainder of the molecule (e.g., M1-H-A-M2, where M1 and M2 are portionsof the molecule, H is a heteroatom and A is an alkylene). Unless statedotherwise specifically in the specification, a heteroalkylene group isoptionally substituted. Exemplary heteroalkylene groups include ethyleneoxide (e.g., polyethylene oxide) and the “C” linking group illustratedbelow:

Multimers of the above C-linker are included in various embodiments ofheteroalkylene linkers.

“Heteroalkenylene” is a heteroalkylene, as defined above, comprising atleast one carbon-carbon double bond. Unless stated otherwisespecifically in the specification, a heteroalkenylene group isoptionally substituted.

“Heteroalkynylene” is a heteroalkylene comprising at least onecarbon-carbon triple bond. Unless stated otherwise specifically in thespecification, a heteroalkynylene group is optionally substituted.

“Heteroatomic” in reference to a “heteroatomic linker” refers to alinker group consisting of one or more heteroatoms. Exemplaryheteroatomic linkers include single atoms selected from the groupconsisting of O, N, P and S, and multiple heteroatoms for example alinker having the formula —P(O⁻)(═O)O— or —OP(O⁻)(═O)O— and multimersand combinations thereof.

“Phosphate” refers to the —OP(═O)(R_(a))R_(b) group, wherein R_(a) isOH, O⁻ or OR_(c); and R_(b) is OH, O⁻, OR_(c), a thiophosphate group ora further phosphate group, wherein R_(c) is a counter ion (e.g., Na+ andthe like).

“Phosphoalkyl” refers to the —OP(═O)(R_(a))R_(b) group, wherein R_(a) isOH, O⁻ or OR_(c); and R_(b) is —Oalkyl, wherein R_(c) is a counter ion(e.g., Na+ and the like). Unless stated otherwise specifically in thespecification, a phosphoalkyl group is optionally substituted. Forexample, in certain embodiments, the —Oalkyl moiety in a phosphoalkylgroup is optionally substituted with one or more of hydroxyl, amino,sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl,phosphoalkylether, thiophosphoalkylether or —OP(═R_(a))(R_(b))R_(c),wherein each of R_(a), R_(b) and R_(c) is as defined for compounds ofstructure (I).

“Phosphoalkylether” refers to the —OP(═O)(R_(a))R_(b) group, whereinR_(a) is OH, O⁻ or OR_(c); and R_(b) is —Oalkylether, wherein R_(c) is acounter ion (e.g., Na+ and the like). Unless stated otherwisespecifically in the specification, a phosphoalkylether group isoptionally substituted. For example, in certain embodiments, the—Oalkylether moiety in a phosphoalkylether group is optionallysubstituted with one or more of hydroxyl, amino, sulfhydryl, phosphate,thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether,thiophosphoalkylether or —OP(═R_(a))(R_(b))R_(c), wherein each of R_(a),R_(b) and R_(c) is as defined for compounds of structure (I).

“Thiophosphate” refers to the —OP(═R_(a))(R_(b))R_(c) group, whereinR_(a) is O or S, R_(b) is OH, O⁻, S⁻, OR_(d) or SR_(d); and R_(c) is OH,SH, O⁻, S⁻, OR_(d), SR_(d), a phosphate group or a further thiophosphategroup, wherein R_(d) is a counter ion (e.g., Na+ and the like) andprovided that: i) R_(a) is S; ii) R_(b) is S⁻ or SR_(d); iii)R_(c) isSH, S⁻ or SR_(d); or iv) a combination of i), ii) and/or iii).

“Thiophosphoalkyl” refers to the —OP(═R_(a))(R_(b))R_(c) group, whereinR_(a) is O or S, R_(b) is OH, O⁻, S⁻, OR_(d) or SR_(d); and R_(c) is—Oalkyl, wherein R_(d) is a counter ion (e.g., Na+ and the like) andprovided that: i) R_(a) is S; ii) R_(b) is S⁻ or SR_(d); or iii)R_(a) isS and R_(b) is S⁻ or SR_(d). Unless stated otherwise specifically in thespecification, a thiophosphoalkyl group is optionally substituted. Forexample, in certain embodiments, the —Oalkyl moiety in athiophosphoalkyl group is optionally substituted with one or more ofhydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl,thiophosphoalkyl, phosphoalkylether, thiophosphoalkylether or—OP(═R_(a))(R_(b))R_(c), wherein each of R_(a), R_(b) and R_(c) is asdefined for compounds of structure (I).

“Thiophosphoalkylether” refers to the —OP(═R_(a))(R_(b))R_(c) group,wherein R_(a) is O or S, R_(b) is OH, O⁻, S⁻, OR_(d) or SR_(d); andR_(c) is —Oalkylether, wherein R_(d) is a counter ion (e.g., Na+ and thelike) and provided that: i) R_(a) is S; ii) R_(b) is S⁻ or SR_(d); oriii)R_(a) is S and R_(b) is S⁻ or SR_(d). Unless stated otherwisespecifically in the specification, a thiophosphoalkylether group isoptionally substituted. For example, in certain embodiments, the—Oalkylether moiety in a thiophosphoalkyl group is optionallysubstituted with one or more of hydroxyl, amino, sulfhydryl, phosphate,thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether,thiophosphoalkylether or —OP(═R_(a))(R_(b))R_(c), wherein each of R_(a),R_(b) and R_(c) is as defined for compounds of structure (I).

“Carbocyclic” refers to a stable 3- to 18-membered aromatic ornon-aromatic ring comprising 3 to 18 carbon atoms. Unless statedotherwise specifically in the specification, a carbocyclic ring may be amonocyclic, bicyclic, tricyclic or tetracyclic ring system, which mayinclude fused or bridged ring systems, and may be partially or fullysaturated. Non-aromatic carbocyclyl radicals include cycloalkyl, whilearomatic carbocyclyl radicals include aryl. Unless stated otherwisespecifically in the specification, a carbocyclic group is optionallysubstituted.

“Cycloalkyl” refers to a stable non-aromatic monocyclic or polycycliccarbocyclic ring, which may include fused or bridged ring systems,having from three to fifteen carbon atoms, preferably having from threeto ten carbon atoms, and which is saturated or unsaturated and attachedto the rest of the molecule by a single bond. Monocyclic cyclocalkylsinclude, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptly, and cyclooctyl. Polycyclic cycloalkyls include, forexample, adamantyl, norbornyl, decalinyl,7,7-dimethyl-bicyclo-[2.2.1]heptanyl, and the like. Unless statedotherwise specifically in the specification, a cycloalkyl group isoptionally substituted.

“Aryl” refers to a ring system comprising at least one carbocyclicaromatic ring. In some embodiments, an aryl comprises from 6 to 18carbon atoms. The aryl ring may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems. Aryls include, but are not limited to, aryls derived fromaceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene,indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene,and triphenylene. Unless stated otherwise specifically in thespecification, an aryl group is optionally substituted.

“Heterocyclic” refers to a stable 3- to 18-membered aromatic ornon-aromatic ring comprising one to twelve carbon atoms and from one tosix heteroatoms selected from the group consisting of nitrogen, oxygenand sulfur. Unless stated otherwise specifically in the specification,the heterocyclic ring may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems; and the nitrogen, carbon or sulfur atoms in the heterocyclicring may be optionally oxidized; the nitrogen atom may be optionallyquaternized; and the heterocyclic ring may be partially or fullysaturated. Examples of aromatic heterocyclic rings are listed below inthe definition of heteroaryls (i.e., heteroaryl being a subset ofheterocyclic). Examples of non-aromatic heterocyclic rings include, butare not limited to, dioxolanyl, thienyl[1,3]dithianyl,decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl,isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl,2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl,piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl,pyrazolopyrimidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl,trioxanyl, trithianyl, triazinanyl, tetrahydropyranyl, thiomorpholinyl,thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl.Unless stated otherwise specifically in the specification, aheterocyclic group is optionally substituted.

“Heteroaryl” refers to a 5- to 14-membered ring system comprising one tothirteen carbon atoms, one to six heteroatoms selected from the groupconsisting of nitrogen, oxygen and sulfur, and at least one aromaticring. For purposes of certain embodiments of this invention, theheteroaryl radical may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems; and the nitrogen, carbon or sulfur atoms in the heteroarylradical may be optionally oxidized; the nitrogen atom may be optionallyquaternized. Examples include, but are not limited to, azepinyl,acridinyl, benzimidazolyl, benzthiazolyl, benzindolyl, benzodioxolyl,benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl,benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl,benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl,benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl(benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl,benzoxazolinonyl, benzimidazolthionyl, carbazolyl, cinnolinyl,dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl,imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl,isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl,oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl,1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl,1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl,phthalazinyl, pteridinyl, pteridinonyl, purinyl, pyrrolyl, pyrazolyl,pyridinyl, pyridinonyl, pyrazinyl, pyrimidinyl, pryrimidinonyl,pyridazinyl, pyrrolyl, pyrido[2,3-d]pyrimidinonyl, quinazolinyl,quinazolinonyl, quinoxalinyl, quinoxalinonyl, quinolinyl, isoquinolinyl,tetrahydroquinolinyl, thiazolyl, thiadiazolyl,thieno[3,2-d]pyrimidin-4-onyl, thieno[2,3-d]pyrimidin-4-onyl, triazolyl,tetrazolyl, triazinyl, and thiophenyl (i.e. thienyl). Unless statedotherwise specifically in the specification, a heteroaryl group isoptionally sub stituted.

“Fused” refers to a ring system comprising at least two rings, whereinthe two rings share at least one common ring atom, for example twocommon ring atoms. When the fused ring is a heterocyclyl ring or aheteroaryl ring, the common ring atom(s) may be carbon or nitrogen.Fused rings include bicyclic, tricyclic, tertracyclic, and the like.

The term “substituted” used herein means any of the above groups (e.g.,alkyl, alkylene, alkenylene, alkynylene, heteroalkylene,heteroalkenylene, heteroalkynylene, alkoxy, alkylether,alkoxyalkylether, heteroalkyl, heteroalkoxy, phosphoalkyl,phosphoalkylether, thiophosphoalkyl, thiophosphoalkylether, carbocyclic,cycloalkyl, aryl, heterocyclic and/or heteroaryl) wherein at least onehydrogen atom (e.g., 1, 2, 3 or all hydrogen atoms) is replaced by abond to a non-hydrogen atoms such as, but not limited to: a halogen atomsuch as F, Cl, Br, and I; an oxygen atom in groups such as hydroxylgroups, alkoxy groups, and ester groups; a sulfur atom in groups such asthiol groups, thioalkyl groups, sulfone groups, sulfonyl groups, andsulfoxide groups; a nitrogen atom in groups such as amines, amides,alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines,N-oxides, imides, and enamines; a silicon atom in groups such astrialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups,and triarylsilyl groups; and other heteroatoms in various other groups.“Substituted” also means any of the above groups in which one or morehydrogen atoms are replaced by a higher-order bond (e.g., a double- ortriple-bond) to a heteroatom such as oxygen in oxo, carbonyl, carboxyl,and ester groups; and nitrogen in groups such as imines, oximes,hydrazones, and nitriles. For example, “substituted” includes any of theabove groups in which one or more hydrogen atoms are replaced with—NR_(g)R_(h), —NR_(g)C(═O)R_(h), —NR_(g)C(═O)NR_(g)R_(h),—NR_(g)C(═O)OR_(h), —NR_(g)SO₂R_(h), —OC(═O)NR_(g)R_(h), —OR_(g),—SR_(g), —SOR_(g), —SO₂R_(g), —OSO₂R_(g), —SO₂OR_(g), ═NSO₂R_(g), and—SO₂NR_(g)R_(h). “Substituted also means any of the above groups inwhich one or more hydrogen atoms are replaced with —C(═O)R_(g),—C(═O)OR_(g), —C(═O)NR_(g)R_(h), —CH₂SO₂R_(g), —CH₂SO₂NR_(g)R_(h). Inthe foregoing, R_(g) and R_(h) are the same or different andindependently hydrogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl,aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl,N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/orheteroarylalkyl. “Substituted” further means any of the above groups inwhich one or more hydrogen atoms are replaced by a bond to an amino,cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkoxy,alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl,haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl,N-heteroaryl and/or heteroarylalkyl group. In some embodiments, theoptional substituent is —OP(═R_(a))(R_(b))R_(c), wherein each of R_(a),R_(b) and R_(c) is as defined for compounds of structure (I). Inaddition, each of the foregoing substituents may also be optionallysubstituted with one or more of the above substituents.

“Conjugation” refers to the overlap of one p-orbital with anotherp-orbital across an intervening sigma bond. Conjugation may occur incyclic or acyclic compounds. A “degree of conjugation” refers to theoverlap of at least one p-orbital with another p-orbital across anintervening sigma bond. For example, 1, 3-butadine has one degree ofconjugation, while benzene and other aromatic compounds typically havemultiple degrees of conjugation. Fluorescent and colored compoundstypically comprise at least one degree of conjugation.

“Fluorescent” refers to a molecule which is capable of absorbing lightof a particular frequency and emitting light of a different frequency.Fluorescence is well-known to those of ordinary skill in the art.

“Colored” refers to a molecule which absorbs light within the coloredspectrum (i.e., red, yellow, blue and the like).

A “linker” refers to a contiguous chain of at least one atom, such ascarbon, oxygen, nitrogen, sulfur, phosphorous and combinations thereof,which connects a portion of a molecule to another portion of the samemolecule or to a different molecule, moiety or solid support (e.g.,microparticle). Linkers may connect the molecule via a covalent bond orother means, such as ionic or hydrogen bond interactions.

The term “biomolecule” refers to any of a variety of biologicalmaterials, including nucleic acids, carbohydrates, amino acids,polypeptides, glycoproteins, hormones, aptamers and mixtures thereof

More specifically, the term is intended to include, without limitation,RNA

DNA, oligonucleotides, modified or derivatized nucleotides, enzymes,receptors, prions, receptor ligands (including hormones), antibodies,antigens, and toxins, as well as bacteria, viruses, blood cells, andtissue cells. The visually detectable biomolecules of the invention(e.g., compounds of structure (I) having a biomolecule linked thereto)are prepared, as further described herein, by contacting a biomoleculewith a compound having a reactive group that enables attachment of thebiomolecule to the compound via any available atom or functional group,such as an amino, hydroxy, carboxyl, or sulfhydryl group on thebiomolecule.

A “reactive group” is a moiety capable of reacting with a secondreactive groups (e.g., a “complementary reactive group”) to form one ormore covalent bonds, for example by a displacement, oxidation,reduction, addition or cycloaddition reaction. Exemplary reactive groupsare provided in Table 1, and include for example, nucleophiles,electrophiles, dienes, dienophiles, aldehyde, oxime, hydrazone, alkyne,amine, azide, acylazide, acylhalide, nitrile, nitrone, sulfhydryl,disulfide, sulfonyl halide, isothiocyanate, imidoester, activated ester,ketone, α,β-unsaturated carbonyl, alkene, maleimide, α-haloimide,epoxide, aziridine, tetrazine, tetrazole, phosphine, biotin, thiiraneand the like.

The terms “visible” and “visually detectable” are used herein to referto substances that are observable by visual inspection, without priorillumination, or chemical or enzymatic activation. Such visuallydetectable substances absorb and emit light in a region of the spectrumranging from about 300 to about 900 nm. Preferably, such substances areintensely colored, preferably having a molar extinction coefficient ofat least about 40,000, more preferably at least about 50,000, still morepreferably at least about 60,000, yet still more preferably at leastabout 70,000, and most preferably at least about 80,000 M⁻¹cm⁻¹. Thecompounds of the invention may be detected by observation with the nakedeye, or with the aid of an optically based detection device, including,without limitation, absorption spectrophotometers, transmission lightmicroscopes, digital cameras and scanners. Visually detectablesubstances are not limited to those which emit and/or absorb light inthe visible spectrum. Substances which emit and/or absorb light in theultraviolet (UV) region (about 10 nm to about 400 nm), infrared (IR)region (about 700 nm to about 1 mm), and substances emitting and/orabsorbing in other regions of the electromagnetic spectrum are alsoincluded with the scope of “visually detectable” substances.

For purposes of embodiments of the invention, the term “photostablevisible dye” refers to a chemical moiety that is visually detectable, asdefined hereinabove, and is not significantly altered or decomposed uponexposure to light. Preferably, the photostable visible dye does notexhibit significant bleaching or decomposition after being exposed tolight for at least one hour. More preferably, the visible dye is stableafter exposure to light for at least 12 hours, still more preferably atleast 24 hours, still yet more preferably at least one week, and mostpreferably at least one month. Nonlimiting examples of photostablevisible dyes suitable for use in the compounds and methods of theinvention include azo dyes, thioindigo dyes, quinacridone pigments,dioxazine, phthalocyanine, perinone, diketopyrrolopyrrole,quinophthalone, and truarycarbonium.

As used herein, the term “perylene derivative” is intended to includeany substituted perylene that is visually detectable. However, the termis not intended to include perylene itself. The terms “anthracenederivative”, “naphthalene derivative”, and “pyrene derivative” are usedanalogously. In some preferred embodiments, a derivative (e.g.,perylene, pyrene, anthracene or naphthalene derivative) is an imide,bisimide or hydrazamimide derivative of perylene, anthracene,naphthalene, or pyrene.

The visually detectable molecules of various embodiments of theinvention are useful for a wide variety of analytical applications, suchas biochemical and biomedical applications, in which there is a need todetermine the presence, location, or quantity of a particular analyte(e.g., biomolecule). In another aspect, therefore, the inventionprovides a method for visually detecting a biomolecule, comprising: (a)providing a biological system with a visually detectable biomoleculecomprising the compound of structure (I) linked to a biomolecule; and(b) detecting the biomolecule by its visible properties. For purposes ofthe invention, the phrase “detecting the biomolecule by its visibleproperties” means that the biomolecule, without illumination or chemicalor enzymatic activation, is observed with the naked eye, or with the aidof a optically based detection device, including, without limitation,absorption spectrophotometers, transmission light microscopes, digitalcameras and scanners. A densitometer may be used to quantify the amountof visually detectable biomolecule present. For example, the relativequantity of the biomolecule in two samples can be determined bymeasuring relative optical density. If the stoichiometry of dyemolecules per biomolecule is known, and the extinction coefficient ofthe dye molecule is known, then the absolute concentration of thebiomolecule can also be determined from a measurement of opticaldensity. As used herein, the term “biological system” is used to referto any solution or mixture comprising one or more biomolecules inaddition to the visually detectable biomolecule. Nonlimiting examples ofsuch biological systems include cells, cell extracts, tissue samples,electrophoretic gels, assay mixtures, and hybridization reactionmixtures.

“Solid support” refers to any solid substrate known in the art forsolid-phase support of molecules, for example a “microparticle” refersto any of a number of small particles useful for attachment to compoundsof the invention, including, but not limited to, glass beads, magneticbeads, polymeric beads, nonpolymeric beads, and the like. In certainembodiments, a microparticle comprises polystyrene beads.

A “solid support reside” refers to the functional group remainingattached to a molecule when the molecule is cleaved from the solidsupport. Solid support residues are known in the art and can be easilyderived based on the structure of the solid support and the grouplinking the molecule thereto.

A “targeting moiety” is a moiety that selectively binds or associateswith a particular target, such as an analyte molecule. “Selectively”binding or associating means a targeting moiety preferentiallyassociates or binds with the desired target relative to other targets.In some embodiments the compounds disclosed herein include linkages totargeting moieties for the purpose of selectively binding or associatingthe compound with an analyte of interest (i.e., the target of thetargeting moiety), thus allowing detection of the analyte. Exemplarytargeting moieties include, but are not limited to, antibodies,antigens, nucleic acid sequences, enzymes, proteins, cell surfacereceptor antagonists, and the like. In some embodiments, the targetingmoiety is a moiety, such as an antibody, that selectively binds orassociates with a target feature on or in a cell, for example a targetfeature on a cell membrane or other cellular structure, thus allowingfor detection of cells of interest. Small molecules that selectivelybind or associate with a desired analyte are also contemplated astargeting moieties in certain embodiments. One of skill in the art willunderstand other analytes, and the corresponding targeting moiety, thatwill be useful in various embodiments.

“Base pairing moiety” refers to a heterocyclic moiety capable ofhybridizing with a complementary heterocyclic moiety via hydrogen bonds(e.g., Watson-Crick base pairing). Base pairing moieties include naturaland unnatural bases. Non-limiting examples of base pairing moieties areRNA and DNA bases such adenosine, guanosine, thymidine, cytosine anduridine and analogues thereof.

Embodiments of the invention disclosed herein are also meant toencompass all compounds of structure (I) or (II) beingisotopically-labelled by having one or more atoms replaced by an atomhaving a different atomic mass or mass number. Examples of isotopes thatcan be incorporated into the disclosed compounds include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, andiodine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³¹P,³²P, ³⁵S, ¹⁸F, ³⁶Cl, ¹²³I, and ¹²⁵I, respectively.

Isotopically-labeled compounds of structure (I) or (II) can generally beprepared by conventional techniques known to those skilled in the art orby processes analogous to those described below and in the followingExamples using an appropriate isotopically-labeled reagent in place ofthe non-labeled reagent previously employed.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

“Optional” or “optionally” means that the subsequently described eventor circumstances may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not. For example, “optionally substituted alkyl” means that thealkyl group may or may not be substituted and that the descriptionincludes both substituted alkyl groups and alkyl groups having nosubstitution.

“Salt” includes both acid and base addition salts.

“Acid addition salt” refers to those salts which are formed withinorganic acids such as, but not limited to, hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and thelike, and organic acids such as, but not limited to, acetic acid,2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid,aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoicacid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproicacid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamicacid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonicacid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid,galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid,glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid,glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid,lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid,malonic acid, mandelic acid, methanesulfonic acid, mucic acid,naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid,1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid,oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamicacid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid,stearic acid, succinic acid, tartaric acid, thiocyanic acid,p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and thelike.

“Base addition salt” refers to those salts which are prepared fromaddition of an inorganic base or an organic base to the free acid. Saltsderived from inorganic bases include, but are not limited to, sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum salts and the like. Salts derived from organic basesinclude, but are not limited to, salts of primary, secondary, andtertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines and basic ion exchange resins, such asammonia, isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, diethanolamine, ethanolamine, deanol,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Particularly preferred organic bases are isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, cholineand caffeine.

Crystallizations may produce a solvate of the compounds describedherein. Embodiments of the present invention include all solvates of thedescribed compounds. As used herein, the term “solvate” refers to anaggregate that comprises one or more molecules of a compound of theinvention with one or more molecules of solvent. The solvent may bewater, in which case the solvate may be a hydrate. Alternatively, thesolvent may be an organic solvent. Thus, the compounds of the presentinvention may exist as a hydrate, including a monohydrate, dihydrate,hemihydrate, sesquihydrate, trihydrate, tetrahydrate and the like, aswell as the corresponding solvated forms. The compounds of the inventionmay be true solvates, while in other cases the compounds of theinvention may merely retain adventitious water or another solvent or bea mixture of water plus some adventitious solvent.

Embodiments of the compounds of the invention (e.g., compounds ofstructure I or II), or their salts, tautomers or solvates may containone or more asymmetric centers and may thus give rise to enantiomers,diastereomers, and other stereoisomeric forms that may be defined, interms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or (L)-for amino acids. Embodiments of the present invention are meant toinclude all such possible isomers, as well as their racemic andoptically pure forms. Optically active (+) and (−), (R)- and (S)-, or(D)- and (L)-isomers may be prepared using chiral synthons or chiralreagents, or resolved using conventional techniques, for example,chromatography and fractional crystallization. Conventional techniquesfor the preparation/isolation of individual enantiomers include chiralsynthesis from a suitable optically pure precursor or resolution of theracemate (or the racemate of a salt or derivative) using, for example,chiral high pressure liquid chromatography (HPLC). When the compoundsdescribed herein contain olefinic double bonds or other centers ofgeometric asymmetry, and unless specified otherwise, it is intended thatthe compounds include both E and Z geometric isomers. Likewise, alltautomeric forms are also intended to be included.

A “stereoisomer” refers to a compound made up of the same atoms bondedby the same bonds but having different three-dimensional structures,which are not interchangeable. The present invention contemplatesvarious stereoisomers and mixtures thereof and includes “enantiomers”,which refers to two stereoisomers whose molecules are nonsuperimposeablemirror images of one another.

A “tautomer” refers to a proton shift from one atom of a molecule toanother atom of the same molecule. The present invention includestautomers of any said compounds. Various tautomeric forms of thecompounds are easily derivable by those of ordinary skill in the art.

The chemical naming protocol and structure diagrams used herein are amodified form of the I.U.P.A.C. nomenclature system, using the ACD/NameVersion 9.07 software program and/or ChemDraw Ultra Version 11.0software naming program (CambridgeSoft). Common names familiar to one ofordinary skill in the art are also used.

As noted above, in one embodiment of the present invention, compoundsuseful as fluorescent and/or colored dyes in various analytical methodsare provided. In other embodiments, compounds useful as syntheticintermediates for preparation of compounds useful as fluorescent and/orcolored dyes are provided. In general terms, embodiments of the presentinvention are directed to dimers and higher polymers of fluorescentand/or colored moieties. The fluorescent and or colored moieties arelinked by a linking moiety. Without wishing to be bound by theory, it isbelieved the linker helps to maintain sufficient spatial distancebetween the fluorescent and/or colored moieties such that intramolecularquenching is reduced or eliminated, thus resulting in a dye compoundhaving a high molar “brightness” (e.g., high fluorescence emission).

Accordingly, in some embodiments the compounds have the followingstructure (A):

wherein L is a linker sufficient to maintain spatial separation betweenone or more (e.g., each) M group so that intramolecular quenching isreduced or eliminated, and R¹, R², R³, L¹, L², L³ and n are as definedfor structure (I). In some embodiments of structure (A), L is a linkercomprising one or more ethylene glycol or polyethylene glycol moieties.

In other embodiments is provided a compound having the followingstructure (I):

or a stereoisomer, salt or tautomer thereof, wherein:

M is, at each occurrence, independently a moiety comprising two or morecarbon-carbon double bonds and at least one degree of conjugation;

L¹ is at each occurrence, independently either: i) an optional alkylene,alkenylene, alkynylene, heteroalkylene, heteroalkenylene,heteroalkynylene or heteroatomic linker; or ii) a linker comprising afunctional group capable of formation by reaction of two complementaryreactive groups;

L² and L³ are, at each occurrence, independently an optional alkylene,alkenylene, alkynylene, heteroalkylene, heteroalkenylene,heteroalkynylene or heteroatomic linker;

L⁴ is, at each occurrence, independently a heteroalkylene,heteroalkenylene or heteroalkynylene linker of greater than three atomsin length, wherein the heteroatoms in the heteroalkylene,heteroalkenylene and heteroalkynylene linker are selected from O, N andS;

R¹ is, at each occurrence, independently H, alkyl or alkoxy;

R² and R³ are each independently H, OH, SH, alkyl, alkoxy, alkylether,heteroalkyl, —OP(═R_(a))(R_(b))R_(c), Q or L′;

R⁴ is, at each occurrence, independently OH, SH, O⁻, S⁻, OR_(a) orSR_(d);

R⁵ is, at each occurrence, independently oxo, thioxo or absent;

R_(a) is O or S;

R_(b) is OH, SH, O⁻, S⁻, OR_(d) or SR_(d);

R_(c) is OH, SH, O⁻, S⁻, OR_(a), OL′, SR_(d), alkyl, alkoxy,heteroalkyl, heteroalkoxy, alkylether, alkoxyalkylether, phosphate,thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether orthiophosphoalkylether;

R_(d) is a counter ion;

Q is, at each occurrence, independently a moiety comprising a reactivegroup, or protected analogue thereof, capable of forming a covalent bondwith an analyte molecule, a targeting moiety, a solid support or acomplementary reactive group Q′;

L′ is, at each occurrence, independently a linker comprising a covalentbond to Q, a linker comprising a covalent bond to a targeting moiety, alinker comprising a covalent bond to an analyte molecule, a linkercomprising a covalent bond to a solid support, a linker comprising acovalent bond to a solid support residue, a linker comprising a covalentbond to a nucleoside or a linker comprising a covalent bond to a furthercompound of structure (I);

m is, at each occurrence, independently an integer of zero or greater,provided that at least one occurrence of m is an integer of one orgreater; and

n is an integer of one or greater.

In different embodiments of the compound of structure (I):

M is, at each occurrence, independently a moiety comprising two or morecarbon-carbon double bonds and at least one degree of conjugation;

L¹ is at each occurrence, independently either: i) an optional alkylene,alkenylene, alkynylene, heteroalkylene, heteroalkenylene,heteroalkynylene or heteroatomic linker; or ii) a linker comprising afunctional group capable of formation by reaction of two complementaryreactive groups;

L² and L³ are, at each occurrence, independently an optional alkylene,alkenylene, alkynylene, heteroalkylene, heteroalkenylene,heteroalkynylene or heteroatomic linker;

L⁴ is, at each occurrence, independently a heteroalkylene,heteroalkenylene or heteroalkynylene linker of greater than three atomsin length, wherein the heteroatoms in the heteroalkylene,heteroalkenylene and heteroalkynylene linker are selected from O, N andS;

R¹ is, at each occurrence, independently H, alkyl or alkoxy;

R² and R³ are each independently H, OH, SH, alkyl, alkoxy, alkylether,—OP(═R_(a))(R_(b))R_(c), Q, a linker comprising a covalent bond to Q, alinker comprising a covalent bond to an analyte molecule, a linkercomprising a covalent bond to a solid support or a linker comprising acovalent bond to a further compound of structure (I), wherein: R_(a) isO or S; R_(b) is OH, SH, O⁻, S⁻, OR_(d) or SR_(d); R_(c) is OH, SH, O⁻,S⁻, OR_(d), SR_(d), alkyl, alkoxy, alkylether, alkoxyalkylether,phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl,phosphoalkylether or thiophosphoalkylether; and R_(d) is a counter ion;

R⁴ is, at each occurrence, independently OH, SH, O⁻, S⁻, OR_(a) orSR_(d);

R⁵ is, at each occurrence, independently oxo, thioxo or absent;

Q is, at each occurrence, independently a moiety comprising a reactivegroup capable of forming a covalent bond with an analyte molecule, asolid support or a complementary reactive group Q′;

m is, at each occurrence, independently an integer of zero or greater,provided that at least one occurrence of m is an integer of one orgreater; and

n is an integer of one or greater.

The various linkers and substituents (e.g., M, Q, R¹, R², R³, R_(c) L¹,L², L³ and L⁴) in the compound of structure (I) are optionallysubstituted with one more substituent. For example, in some embodimentsthe optional substituent is selected to optimize the water solubility orother property of the compound of structure (I). In certain embodiments,each alkyl, alkoxy, alkylether, alkoxyalkylether, phosphoalkyl,thiophosphoalkyl, phosphoalkylether and thiophosphoalkylether in thecompound of structure (I) is optionally substituted with one moresubstituent selected from the group consisting of hydroxyl, alkoxy,alkylether, alkoxyalkylether, sulfhydryl, amino, alkylamino, carboxyl,phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl,phosphoalkylether and thiophosphoalkylether. In certain embodiments theoptional substituent is —OP(═R_(a))(R_(b))R_(c), where R_(a), R_(b) andR, are as defined for the compound of structure (I).

In some embodiments, L¹ is at each occurrence, independently an optionalalkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene,heteroalkynylene or heteroatomic linker. In other embodiments, L¹ is ateach occurrence, independently a linker comprising a functional groupcapable of formation by reaction of two complementary reactive groups,for example a Q group.

In some embodiments, L⁴ is at each occurrence, independently aheteroalkylene linker. In other more specific embodiments, L⁴ is at eachoccurrence, independently an alkylene oxide linker. For example, in someembodiments L⁴ is polyethylene oxide, and the compound has the followingstructure (IA):

wherein z is an integer from 2 to 100. In some embodiments of (IA), z isan integer from 2-30, for example from about 20 to 25, or about 23. Insome embodiments, z is an integer from 2 to 10, for example from 3 to 6.In some embodiments, z is 3. In some embodiments, z is 4. In someembodiments, z is 5. In some embodiments, z is 6.

The optional linker L¹ can be used as a point of attachment of the Mmoiety to the remainder of the compound. For example, in someembodiments a synthetic precursor to the compound of structure (I) isprepared, and the M moiety is attached to the synthetic precursor usingany number of facile methods known in the art, for example methodsreferred to as “click chemistry.” For this purpose any reaction which israpid and substantially irreversible can be used to attach M to thesynthetic precursor to form a compound of structure (I). Exemplaryreactions include the copper catalyzed reaction of an azide and alkyneto form a triazole (Huisgen 1, 3-dipolar cycloaddition), reaction of adiene and dienophile (Diels-Alder), strain-promoted alkyne-nitronecycloaddition, reaction of a strained alkene with an azide, tetrazine ortetrazole, alkene and azide [3+2] cycloaddition, alkene and tetrazineinverse-demand Diels-Alder, alkene and tetrazole photoreaction andvarious displacement reactions, such as displacement of a leaving groupby nucleophilic attack on an electrophilic atom. Exemplary displacementreactions include reaction of an amine with: an activated ester; anN-hydroxysuccinimide ester; an isocyanate; an isothioscyanate or thelike. In some embodiments the reaction to form L¹ may be performed in anaqueous environment.

Accordingly, in some embodiments L¹ is at each occurrence a linkercomprising a functional group capable of formation by reaction of twocomplementary reactive groups, for example a functional group which isthe product of one of the foregoing “click” reactions. In variousembodiments, for at least one occurrence of L, the functional group canbe formed by reaction of an aldehyde, oxime, hydrazone, alkyne, amine,azide, acylazide, acylhalide, nitrile, nitrone, sulfhydryl, disulfide,sulfonyl halide, isothiocyanate, imidoester, activated ester (e.g.,N-hydroxysuccinimide ester), ketone, α,β-unsaturated carbonyl, alkene,maleimide, a-haloimide, epoxide, aziridine, tetrazine, tetrazole,phosphine, biotin or thiirane functional group with a complementaryreactive group. For example, reaction of an amine with anN-hydroxysuccinimide ester or isothiocyanate.

In other embodiments, for at least one occurrence of L, the functionalgroup can be formed by reaction of an alkyne and an azide. In otherembodiments, for at least one occurrence of L, the functional group canbe formed by reaction of an amine (e.g., primary amine) and anN-hydroxysuccinimide ester or isothiocyanate.

In more embodiments, for at least one occurrence of L, the functionalgroup comprises an alkene, ester, amide, thioester, disulfide,carbocyclic, heterocyclic or heteroaryl group. In more embodiments, forat least one occurrence of L¹, the functional group comprises an alkene,ester, amide, thioester, thiourea, disulfide, carbocyclic, heterocyclicor heteroaryl group. In other embodiments, the functional groupcomprises an amide or thiourea. In some more specific embodiments, forat least one occurrence of L¹, L¹ is a linker comprising a triazolylfunctional group. While in other embodiments, for at least oneoccurrence of L, L¹ is a linker comprising an amide or thioureafunctional group.

In still other embodiments, for at least one occurrence of L¹, L¹-M hasthe following structure:

wherein L^(1a) and L^(1b) are each independently optional linkers.

In different embodiments, for at least one occurrence of L¹, L¹-M hasthe following structure:

wherein L^(1a) and L^(1b) are each independently optional linkers.

In various embodiments of the foregoing, L^(1a) or L^(1b), or both, isabsent. In other embodiments, L^(1a) or L^(1b), or both, is present.

In some embodiments L^(1a) and L^(1b), when present, are eachindependently alkylene or heteroalkylene. For example, in someembodiments L^(1a) and L^(1b), when present, independently have one ofthe following structures:

In still other different embodiments of structure (I), L¹ is at eachoccurrence, independently an optional alkylene or heteroalkylene linker.In certain embodiments, L¹ has one of the following structures:

In more embodiments, L² and L³ are, at each occurrence, independentlyC₁-C₆ alkylene, C₂-C₆ alkenylene or C₂-C₆ alkynylene. For example, insome embodiments the compound has the following structure (IB):

wherein:

x¹, x², x³ and x⁴ are, at each occurrence, independently an integer from0 to 6; and

z is an integer from 2 to 100, for example from 3 to 6.

In certain embodiments of the compound of structure (IB), at least oneoccurrence of x¹, x², x³ or x⁴ is 1. In other embodiments, x¹, x², x³and x⁴ are each 1 at each occurrence. In other embodiments, x¹ and x³are each 0 at each occurrence. In some embodiments, x² and x⁴ are each 1at each occurrence. In still other embodiments, x¹ and x³ are each 0 ateach occurrence, and x² and x⁴ are each 1 at each occurrence.

In some more specific embodiments of the compound of structure (IB), L¹,at each occurrence, independently comprises a triazolyl functionalgroup. In some other specific embodiments of the compound of structure(IB), L¹, at each occurrence, independently comprises an amide orthiourea functional group. In other embodiments of the compound ofstructure (IB), L¹, at each occurrence, independently an optionalalkylene or heteroalkylene linker.

In still other embodiments of any of the compounds of structure (I), R⁴is, at each occurrence, independently OH, O⁻ or OR_(d). It is understoodthat “OR_(d)” and “SR_(d)” are intended to refer to O⁻ and S⁻ associatedwith a cation. For example, the disodium salt of a phosphate group maybe represented as:

where R_(d) is sodium (Na⁺).

In other embodiments of any of the compounds of structure (I), R⁵ is, ateach occurrence, oxo.

In some different embodiments of any of the foregoing compounds, R¹ isH.

In other various embodiments, R² and R³ are each independently OH or—OP(═R_(a))(R_(b))R_(c). In some different embodiments, R² or R³ is OHor —OP(═R_(a))(R_(b))R_(c), and the other of R² or R³ is Q or a linkercomprising a covalent bond to Q.

In still more different embodiments of any of the foregoing compounds ofstructure (I), R² and R³ are each independently —OP(═R_(a))(R_(b))R_(c).In some of these embodiments, R_(c) is OL′.

In other embodiments, R² and R³ are each independently—OP(═R_(a))(R_(b))OL′, and L′ is an alkylene or heteroalkylene linkerto: Q, a targeting moiety, an analyte (e.g., analyte molecule), a solidsupport, a solid support residue, a nucleoside or a further compound ofstructure (I).

The linker L′ can be any linker suitable for attaching Q, a targetingmoiety, an analyte (e.g., analyte molecule), a solid support, a solidsupport residue, a nucleoside or a further compound of structure (I) tothe compound of structure (I). Advantageously certain embodimentsinclude use of L′ moieties selected to increase or optimize watersolubility of the compound. In certain embodiments, L′ is aheteroalkylene moiety. In some other certain embodiments, L′ comprisesan alkylene oxide or phosphodiester moiety, or combinations thereof.

In certain embodiments, L′ has the following structure:

wherein:

m″ and n″ are independently an integer from 1 to 10;

R^(e) is H, an electron pair or a counter ion;

L″ is R^(e) or a direct bond or linkage to: Q, a targeting moiety, ananalyte (e.g., analyte molecule), a solid support, a solid supportresidue, a nucleoside or a further compound of structure (I).

In some embodiments, m″ is an integer from 4 to 10, for example 4, 6 or10. In other embodiments n″ is an integer from 3 to 6, for example 3, 4,5 or 6.

In some other embodiments, L″ is an alkylene or heteroalkylene moiety.In some other certain embodiments, L″ comprises an alkylene oxide,phosphodiester moiety, sulfhydryl, disulfide or maleimide moiety orcombinations thereof.

In certain of the foregoing embodiments, the targeting moiety is anantibody or cell surface receptor antagonist.

In other more specific embodiments of any of the foregoing compounds ofstructure (I), R² or R³ has one of the following structures:

Certain embodiments of compounds of structure (I) can be preparedaccording to solid-phase synthetic methods analogous to those known inthe art for preparation of oligonucleotides. Accordingly, in someembodiments, L′ is a linkage to a solid support, a solid support residueor a nucleoside. Solid supports comprising an activated deoxythymidine(dT) group are readily available, and in some embodiments can beemployed as starting material for preparation of compounds of structure(I). Accordingly, in some embodiments R² or R³ has the followingstructure:

One of skill in the art will understand that the dT group depicted aboveis included for ease of synthesis and economic efficiencies only, and isnot required. Other solid supports can be used and would result in adifferent nucleoside or solid support residue being present on L′, orthe nucleoside or solid support residue can be removed or modified postsynthesis.

In still other embodiments, Q is, at each occurrence, independently amoiety comprising a reactive group capable of forming a covalent bondwith an analyte molecule or a solid support. In other embodiments, Q is,at each occurrence, independently a moiety comprising a reactive groupcapable of forming a covalent bond with a complementary reactive groupQ′. For example, in some embodiments, Q′ is present on a furthercompound of structure (I) (e.g., in the R² or R³ position), and Q and Q′comprise complementary reactive groups such that reaction of thecompound of structure (I) and the further compound of structure (I)results in covalently bound dimer of the compound of structure (I).Multimer compounds of structure (I) can also be prepared in an analogousmanner and are included within the scope of embodiments of theinvention.

The type of Q group and connectivity of the Q group to the remainder ofthe compound of structure (I) is not limited, provided that Q comprisesa moiety having appropriate reactivity for forming the desired bond.

In certain embodiments, Q is a moiety which is not susceptible tohydrolysis under aqueous conditions, but is sufficiently reactive toform a bond with a corresponding group on an analyte molecule or solidsupport (e.g., an amine, azide or alkyne).

Certain embodiments of compounds of structure (I) comprise Q groupscommonly employed in the field of bioconjugation. For example in someembodiments, Q comprises a nucleophilic reactive group, an electrophilicreactive group or a cycloaddition reactive group. In some more specificembodiments, Q comprises a sulfhydryl, disulfide, activated ester,isothiocyanate, azide, alkyne, alkene, diene, dienophile, acid halide,sulfonyl halide, phosphine, c-haloamide, biotin, amino or maleimidefunctional group. In some embodiments, the activated ester is anN-succinimide ester, imidoester or polyflourophenyl ester. In otherembodiments, the alkyne is an alkyl azide or acyl azide.

The Q groups can be conveniently provided in protected form to increasestorage stability or other desired properties, and then the protectinggroup removed at the appropriate time for conjugation with, for example,a targeting moiety or analyte. Accordingly, Q groups include “protectedforms” of a reactive group, including any of the reactive groupsdescribed above and in the Table 1 below. A “protected form” of Q refersto a moiety having lower reactivity under predetermined reactionconditions relative to Q, but which can be converted to Q underconditions, which preferably do not degrade or react with other portionsof the compound of structure (I). One of skill in the art can deriveappropriate protected forms of Q based on the particular Q and desiredend use and storage conditions. For example, when Q is SH, a protectedform of Q includes a disulfide, which can be reduce to reveal the SHmoiety using commonly known techniques and reagents.

Exemplary Q moieties are provided in Table I below.

TABLE 1 Exemplary Q Moieties Structure Class

Sulfhydryl

Isothiocyanate

Imidoester

Acyl Azide

Activated Ester

Activated Ester

Activated Ester

Activated Ester

Activated Ester

Activated Ester

Sulfonyl halide

Maleimide

Maleimide

Maleimide

α-haloimide

Disulfide

Phosphine

Azide

Alkyne

Biotin

Diene

Alkene/ dienophile

Alkene/ dienophile —NH₂ Amino

It should be noted that in some embodiments, wherein Q is SH, the SHmoiety will tend to form disulfide bonds with another sulfhydryl group,for example on another compound of structure (I). Accordingly, someembodiments include compounds of structure (I), which are in the form ofdisulfide dimers, the disulfide bond being derived from SH Q groups.

Also included within the scope of certain embodiments are compounds ofstructure (I), wherein one, or both, of R² and R³ comprises a linkage toa further compound of structure (I). For example, wherein one or both ofR² and R³ are —OP(═R_(a))(R_(b))R_(c), and Rc is OL′, and L′ is a linkercomprising a covalent bond to a further compound of structure (I). Suchcompounds can be prepared by preparing a first compound of structure (I)having for example about 10 “M” moieties (i.e., n=9) and having anappropriate “Q” for reaction with a complementary Q′ group on a secondcompound of structure (I). In this manner, compounds of structure (I),having any number of “M” moieties, for example 100 or more, can beprepared without the need for sequentially coupling each monomer.Exemplary embodiments of such compounds of structure (I) have thefollowing structure (I′)

wherein:

each occurrence of R¹, R², R³, R⁴, R⁵, L¹, L², L³, L⁴, M, m and n areindependently as defined for a compound of structure (I);

L″ is a linker comprising a functional group resulting from reaction ofa Q moiety with a corresponding Q′ moiety; and

α is an integer greater than 1, for example from 1 to 100, or 1 to 10.

An exemplary compound of structure (I′) is provided in Example 5. Othercompounds of structure (I′) are derivable by those of ordinary skill inthe art, for example by dimerizing or polymerizing compounds ofstructure (I) provided herein.

In other embodiments, the Q moiety is conveniently masked (e.g.,protected) as a disulfide moiety, which can later be reduced to providean activated Q moiety for binding to a desired analyte molecule ortargeting moiety. For example, the Q moiety may be masked as a disulfidehaving the following structure:

wherein R is an optionally substituted alkyl group. For example, in someembodiments, Q is provided as a disulfide moiety having the followingstructure:

where n is an integer from 1 to 10, for example 6.

In some other embodiments, one of R² or R³ is OH or—OP(═R_(a))(R_(b))R_(c), and the other of R² or R³ is a linkercomprising a covalent bond to an analyte molecule or a linker comprisinga covalent bond to a solid support. For example, in some embodiments theanalyte molecule is a nucleic acid, amino acid or a polymer thereof. Inother embodiments, the analyte molecule is an enzyme, receptor, receptorligand, antibody, glycoprotein, aptamer or prion. In still differentembodiments, the solid support is a polymeric bead or nonpolymeric bead.

The value for m is another variable that can be selected based on thedesired fluorescence and/or color intensity. In some embodiments, m is,at each occurrence, independently an integer from 1 to 10. In otherembodiments, m is, at each occurrence, independently an integer from 1to 5, for example 1, 2, 3, 4 or 5.

In other embodiments, m is, at each occurrence, independently an integergreater than 2, and z is an integer from 3 to 10, for example in someembodiment m is, at each occurrence, independently an integer greaterthan 2, such as 3, 4, 5 or 6, and z is an integer from 3 to 6.

The fluorescence intensity can also be tuned by selection of differentvalues of n. In certain embodiments, n is an integer from 1 to 100. Inother embodiments, n is an integer from 1 to 10. In some embodiments nis 1. In some embodiments n is 2. In some embodiments n is 3. In someembodiments n is 4. In some embodiments n is 5. In some embodiments n is6. In some embodiments n is 7. In some embodiments n is 8. In someembodiments n is 9. In some embodiments n is 10.

M is selected based on the desired optical properties, for example basedon a desired color and/or fluorescence emission wavelength. In someembodiments, M is the same at each occurrence; however, it is importantto note that each occurrence of M need not be an identical M, andcertain embodiments include compounds wherein M is not the same at eachoccurrence. For example, in some embodiments each M is not the same andthe different M moieties are selected to have absorbance and/oremissions for use in fluorescence resonance energy transfer (FRET)methods. For example, in such embodiments the different M moieties areselected such that absorbance of radiation at one wavelength causesemission of radiation at a different wavelength by a FRET mechanism.Exemplary M moieties can be appropriately selected by one of ordinaryskill in the art based on the desired end use. Exemplary M moieties forFRET methods include fluorescein and 5-TAMRA(5-carboxytetramethylrhodamine, succinimidyl ester) dyes.

M may be attached to the remainder of the molecule from any position(i.e., atom) on M. One of skill in the art will recognize means forattaching M to the remainder of molecule. Exemplary methods include the“click” reactions described herein.

In some embodiments, M is a fluorescent or colored moiety. Anyfluorescent and/or colored moiety may be used, for examples those knownin the art and typically employed in colorimetric, UV, and/orfluorescent assays may be used. Examples of M moieties which are usefulin various embodiments of the invention include, but are not limited to:Xanthene derivatives (e.g., fluorescein, rhodamine, Oregon green, eosinor Texas red); Cyanine derivatives (e.g., cyanine, indocarbocyanine,oxacarbocyanine, thiacarbocyanine or merocyanine); Squaraine derivativesand ring-substituted squaraines, including Seta, SeTau, and Square dyes;Naphthalene derivatives (e.g., dansyl and prodan derivatives); Coumarinderivatives; oxadiazole derivatives (e.g., pyridyloxazole,nitrobenzoxadiazole or benzoxadiazole); Anthracene derivatives (e.g.,anthraquinones, including DRAQ5, DRAQ7 and CyTRAK Orange); Pyrenederivatives such as cascade blue; Oxazine derivatives (e.g., Nile red,Nile blue, cresyl violet, oxazine 170); Acridine derivatives (e.g.,proflavin, acridine orange, acridine yellow); Arylmethine derivatives:auramine, crystal violet, malachite green; and Tetrapyrrole derivatives(e.g., porphin, phthalocyanine or bilirubin). Other exemplary M moietiesinclude: Cyanine dyes, xanthate dyes (e.g., Hex, Vic, Nedd, Joe or Tet);Yakima yellow; Redmond red; tamra; texas red and Alexa Fluor® dyes.

In still other embodiments of any of the foregoing, M comprises three ormore aryl or heteroaryl rings, or combinations thereof, for example fouror more aryl or heteroaryl rings, or combinations thereof, or even fiveor more aryl or heteroaryl rings, or combinations thereof. In someembodiments, M comprises six aryl or heteroaryl rings, or combinationsthereof. In further embodiments, the rings are fused. For example insome embodiments, M comprises three or more fused rings, four or morefused rings, five or more fused rings, or even six or more fused rings.

In some embodiments, M is cyclic. For example, in some embodiments M iscarbocyclic. In other embodiment, M is heterocyclic. In still otherembodiments of the foregoing, M, at each occurrence, independentlycomprises an aryl moiety. In some of these embodiments, the aryl moietyis multicyclic. In other more specific examples, the aryl moiety is afused-multicyclic aryl moiety, for example which may comprise at least3, at least 4, or even more than 4 aryl rings.

In other embodiments of any of the foregoing compounds of structure (I),(IA), (IB) or (I′), M, at each occurrence, independently comprises atleast one heteroatom. For example, in some embodiments, the heteroatomis nitrogen, oxygen or sulfur.

In still more embodiments of any of the foregoing, M, at eachoccurrence, independently comprises at least one substituent. Forexample, in some embodiments the substituent is a fluoro, chloro, bromo,iodo, amino, alkylamino, arylamino, hydroxy, sulfhydryl, alkoxy,aryloxy, phenyl, aryl, methyl, ethyl, propyl, butyl, isopropyl, t-butyl,carboxy, sulfonate, amide, or formyl group.

In some even more specific embodiments of the foregoing, M, at eachoccurrence, independently is a dimethylaminostilbene, quinacridone,fluorophenyl-dimethyl-BODIPY, his-fluorophenyl-BODIPY, acridine,terrylene, sexiphenyl, porphyrin, benzopyrene,(fluorophenyl-dimethyl-difluorobora-diaza-indacene)phenyl,(bis-fluorophenyl-difluorobora-diaza-indacene)phenyl, quaterphenyl,bi-benzothiazole, ter-benzothiazole, bi-naphthyl, bi-anthracyl,squaraine, squarylium, 9, 10-ethynylanthracene or ter-naphthyl moiety.In other embodiments, M is, at each occurrence, independentlyp-terphenyl, perylene, azobenzene, phenazine, phenanthroline, acridine,thioxanthrene, chrysene, rubrene, coronene, cyanine, perylene imide, orperylene amide or a derivative thereof. In still more embodiments, M is,at each occurrence, independently a coumarin dye, resorufin dye,dipyrrometheneboron difluoride dye, ruthenium bipyridyl dye, energytransfer dye, thiazole orange dye, polymethine orN-aryl-1,8-naphthalimide dye.

In still more embodiments of any of the foregoing, M at each occurrenceis the same. In other embodiments, each M is different. In still moreembodiments, one or more M is the same and one or more M is different.

In some embodiments, M is pyrene, perylene, perylene monoimide or 6-FAMor a derivative thereof. In some other embodiments, M has one of thefollowing structures:

Although M moieties comprising carboxylic acid groups are depicted inthe anionic form (CO₂) above, one of skill in the art will understandthat this will vary depending on pH, and the protonated form (CO₂H) isincluded in various embodiments.

In some specific embodiments, the compound is a compound selected fromTable 2. The compounds in Table 2 were prepared according to theprocedures set forth in the Examples and their identity confirmed bymass spectrometry.

TABLE 2 Exemplary Compounds of Structure I MW. Found No. Calc. StructureI-1 1364.6 1365.2

I-2 1576.2 1577.3

I-3 1497.4 1497.3

I-4 1841.4 1841.6

I-5 2185.8 2185.9

I-6 2532.2 2530.2

I-7 1789.6 1789.5

I-8 2001.6 2001.6

I-9 2213.5 2213.8

I-10 4481.6 4480.9

I-11 8375.9 8374.3

I-12 TBD

I-13 TBD

I-14 TBD

I-15 TBD

I-16 TBD

I-17 15684.6 15681.5

I-18 TBD

I-19 TBD

I-20 TBD

I-21 TBD

I-22 TBD

I-23 TBD

I-24 TBD

I-25 TBD

I-26 TBD

I-27 TBD

I-28 TBD

I-29 TBD

I-30 TBD

I-31 TBD

I-32 7241.2 7238.2

I-33 TBD

I-34 TBD

I-35 TBD

I-36 TBD

I-37 6997.1 6997.0

I-38 TBD

I-39 TBD

I-40 TBD

I-41 TBD

I-42 TBD

I-43 3103.9 3103.6

I-44 5619.5 5619.8

I-45 15684.6 15681.5

I-46 6997.1 6997.0

I-47 11912.1 11910.1

I-48 9273.9 9272.0

I-49 16252.9 16250.0

I-50 17260.3 17260.0

I-51 TBD

I-52 TBD

I-53 TBD

I-54 TBD

I-55 TBD

I-56 TBD

I-57 TBD

I-58 TBD

I-59 TBD

I-60 TBD

*TBD = to be determined

As used in Table 2 and throughout the application R², R³, m, n and L′have the definitions provided for compounds of structure (I) unlessotherwise indicated, and F, F′ and F″ refer to a fluorescein moietyhaving the following structures, respectively:

“dT” refers to the following structure:

Some embodiments include any of the foregoing compounds, including thespecific compounds provided in Table 2, conjugated to a targetingmoiety, such as an antibody.

The present disclosure generally provides compounds having increasedfluorescence emission relative to earlier known compounds. Accordingly,certain embodiments are directed to a fluorescent compound comprising Yfluorescent moieties M, wherein the fluorescent compound has a peakfluorescence emission upon excitation with a predetermined wavelength ofultraviolet light of at least 85% of Y times greater than the peakfluorescence emission of a single M moiety upon excitation with the samewavelength of ultraviolet light, and wherein Y is an integer of 2 ormore. Fluorescent compounds include compounds which emit a fluorescentsignal upon excitation with light, such as ultraviolet light.

In some embodiments, the fluorescent compound has a peak fluorescenceemission of at least 90% of Y times greater, 95% of Y times greater, 97%of Y times greater or 99% of Y times greater than the peak fluorescenceemission of a single M moiety.

In some embodiments, Y is an integer from 2 to 100, for example 2-10.

In some embodiments, the Y M moiety have, independently, one of thefollowing structures:

wherein

indicates a point of attachment to the fluorescent compound.

In other embodiments, the single M moiety has, independently, one of thefollowing structures:

In more specific embodiments, the fluorescent compound comprises Y Mmoieties, independently having one of the following structures:

wherein

indicates a point of attachment to the fluorescent compound, and thesingle M moiety has the following structures

In other embodiments, the peak fluorescence emission is at a wavelengthranging from about 500 to about 550 nm.

In still more embodiments, the fluorescent compound comprises at leastone ethylene oxide moiety.

Compositions comprising the fluorescent compound of any one of claimsand an analyte are also provided.

The presently disclosed compounds are “tunable,” meaning that by properselection of the variables in any of the foregoing compounds, one ofskill in the art can arrive at a compound having a desired and/orpredetermined molar fluorescence (molar brightness). The tunability ofthe compounds allows the user to easily arrive at compounds having thedesired fluorescence and/or color for use in a particular assay or foridentifying a specific analyte of interest. Although all variables mayhave an effect on the molar fluorescence of the compounds, properselection of M, L⁴, m and n is believed to play an important role in themolar fluorescence of the compounds. Accordingly, in one embodiment isprovided a method for obtaining a compound having a desired molarfluorescence, the method comprising selecting an M moiety having a knownfluorescence, preparing a compound of structure (I) comprising the Mmoiety, and selecting the appropriate variables for L⁴, m and n toarrive at the desired molar fluorescence.

Molar fluorescence in certain embodiments can be expressed in terms ofthe fold increase or decrease relative to the fluorescence emission ofthe parent fluorophore (e.g., monomer). In some embodiments the molarfluorescence of the present compounds is 1.1×, 1.5×, 2×, 3×, 4×, 5×, 6×,7×, 8×, 9×10× or even higher relative to the parent fluorophore. Variousembodiments include preparing compounds having the desired fold increasein fluorescence relative to the parent fluorophore by proper selectionof L⁴, m and n.

For ease of illustration, various compounds comprising phosphorousmoieties (e.g., phosphate and the like) are depicted in the anionicstate (e.g., —OPO(OH)O⁻, —OPO₃ ²⁻). One of skill in the art will readilyunderstand that the charge is dependent on pH and the uncharged (e.g.,protonated or salt, such as sodium or other cation) forms are alsoincluded in the scope of embodiments of the invention.

Compositions comprising any of the foregoing compounds and one or moreanalyte molecules (e.g., biomolecules) are provided in various otherembodiments. In some embodiments, use of such compositions in analyticalmethods for detection of the one or more analyte molecules are alsoprovided.

In still other embodiments, the compounds are useful in variousanalytical methods. For example, in certain embodiments the disclosureprovides a method of staining a sample, the method comprising adding tosaid sample a compound of structure (I), for example wherein one of R²or R³ is a linker comprising a covalent bond to an analyte molecule(e.g., biomolecule) or microparticle, and the other of R² or R³ is H,OH, alkyl, alkoxy, alkylether or —OP(═R_(a))(R_(b))R_(c), in an amountsufficient to produce an optical response when said sample isilluminated at an appropriate wavelength.

In some embodiments of the foregoing methods, R² is a linker comprisinga covalent linkage to an analyte molecule, such as a biomolecule. Forexample, a nucleic acid, amino acid or a polymer thereof (e.g.,polynucleotide or polypeptide). In still more embodiments, thebiomolecule is an enzyme, receptor, receptor ligand, antibody,glycoprotein, aptamer or prion.

In yet other embodiments of the foregoing method, R² is a linkercomprising a covalent linkage to a solid support such as amicroparticle. For example, in some embodiments the microparticle is apolymeric bead or nonpolymeric bead.

In even more embodiments, said optical response is a fluorescentresponse.

In other embodiments, said sample comprises cells, and some embodimentsfurther comprise observing said cells by flow cytometry.

In still more embodiments, the method further comprises distinguishingthe fluorescence response from that of a second fluorophore havingdetectably different optical properties.

In other embodiments, the disclosure provides a method for visuallydetecting an analyte molecule, such as a biomolecule, comprising:

-   -   (a) providing a compound of structure (I), for example, wherein        one of R² or R³ is a linker comprising a covalent bond to the        analyte molecule, and the other of R² or R³ is H, OH, alkyl,        alkoxy, alkylether or —OP(═R_(a))(R_(b))R_(c); and    -   (b) detecting the compound by its visible properties.

In some embodiments the analyte molecule is a nucleic acid, amino acidor a polymer thereof (e.g., polynucleotide or polypeptide). In stillmore embodiments, the analyte molecule is an enzyme, receptor, receptorligand, antibody, glycoprotein, aptamer or prion.

In other embodiments, a method for visually detecting an analytemolecule, such as a biomolecule is provided, the method comprising:

-   -   (a) admixing any of the foregoing compounds with one or more        analyte molecules; and    -   (b) detecting the compound by its visible properties.

In other embodiments is provided a method for visually detecting ananalyte molecule, the method comprising:

-   -   (a) admixing the compound of claim 1, wherein R² or R³ is Q or a        linker comprising a covalent bond to Q, with the analyte        molecule;    -   (b) forming a conjugate of the compound and the analyte        molecule; and    -   (c) detecting the conjugate by its visible properties.

Other exemplary methods include a method for detecting an analyte, themethod comprising:

-   -   (a) providing a compound of structure (I), wherein R² or R³        comprises a linker comprising a covalent bond to a targeting        moiety having specificity for the analyte;    -   (b) admixing the compound and the analyte, thereby associating        the targeting moiety and the analyte; and    -   (c) detecting the compound, for example by its visible or        fluorescent properties.

In certain embodiments of the foregoing method, the analyte is aparticle, such as a cell, and the method includes use of flow cytometry.For example, the compound may be provided with a targeting moiety, suchas an antibody, for selectively associating with the desired cell, thusrendering the cell detectable by any number of techniques, such asvisible or fluorescence detection. Appropriate antibodies can beselected by one of ordinary skill in the art depending on the desiredend use. Exemplary antibodies for use in certain embodiments includeUCHT1 and MOPC-21.

Embodiments of the present compounds thus find utility in any number ofmethods, including, but not limited: cell counting; cell sorting;biomarker detection; quantifying apoptosis; determining cell viability;identifying cell surface antigens; determining total DNA and/or RNAcontent; identifying specific nucleic acid sequences (e.g., as a nucleicacid probe); and diagnosing diseases, such as blood cancers.

In addition to the above methods, embodiments of the compounds ofstructure (I) find utility in various disciplines and methods, includingbut not limited to: imaging in endoscopy procedures for identificationof cancerous and other tissues; single-cell and/or single moleculeanalytical methods, for example detection of polynucleotides with littleor no amplification; cancer imaging, for example by including atargeting moiety, such as an antibody or sugar or other moiety thatpreferentially binds cancer cells, in a compound of structure (I) to;imaging in surgical procedures; binding of histones for identificationof various diseases; drug delivery, for example by replacing the Mmoiety in a compound of structure (I) with an active drug moiety; and/orcontrast agents in dental work and other procedures, for example bypreferential binding of the compound of structure (I) to various floraand/or organisms.

It is understood that any embodiment of the compounds of structure (I),as set forth above, and any specific choice set forth herein for a R¹,R², R³, R⁴, R⁵, L′, L¹, L², L³, L⁴, M, m and/or n variable in thecompounds of structure (I), as set forth above, may be independentlycombined with other embodiments and/or variables of the compounds ofstructure (I) to form embodiments of the invention not specifically setforth above. In addition, in the event that a list of choices is listedfor any particular R¹, R², R³, R⁴, R⁵, L′, L¹, L², L³, L⁴, M, m and/or nvariable in a particular embodiment and/or claim, it is understood thateach individual choice may be deleted from the particular embodimentand/or claim and that the remaining list of choices will be consideredto be within the scope of the invention.

It is understood that in the present description, combinations ofsubstituents and/or variables of the depicted formulae are permissibleonly if such contributions result in stable compounds.

It will also be appreciated by those skilled in the art that in theprocess described herein the functional groups of intermediate compoundsmay need to be protected by suitable protecting groups. Such functionalgroups include hydroxy, amino, mercapto and carboxylic acid. Suitableprotecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl(for example, t-butyldimethylsilyl, t-butyldiphenylsilyl ortrimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitableprotecting groups for amino, amidino and guanidino includet-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protectinggroups for mercapto include —C(O)—R″ (where R″ is alkyl, aryl orarylalkyl), p-methoxybenzyl, trityl and the like. Suitable protectinggroups for carboxylic acid include alkyl, aryl or arylalkyl esters.Protecting groups may be added or removed in accordance with standardtechniques, which are known to one skilled in the art and as describedherein. The use of protecting groups is described in detail in Green, T.W. and P. G. M. Wutz, Protective Groups in Organic Synthesis (1999), 3rdEd., Wiley. As one of skill in the art would appreciate, the protectinggroup may also be a polymer resin such as a Wang resin, Rink resin or a2-chlorotrityl-chloride resin.

Furthermore, all compounds of the invention which exist in free base oracid form can be converted to their salts by treatment with theappropriate inorganic or organic base or acid by methods known to oneskilled in the art. Salts of the compounds of the invention can beconverted to their free base or acid form by standard techniques.

The following Reaction Schemes illustrate exemplary methods of makingcompounds of this invention. It is understood that one skilled in theart may be able to make these compounds by similar methods or bycombining other methods known to one skilled in the art. It is alsounderstood that one skilled in the art would be able to make, in asimilar manner as described below, other compounds of structure (I) notspecifically illustrated below by using the appropriate startingcomponents and modifying the parameters of the synthesis as needed. Ingeneral, starting components may be obtained from sources such as SigmaAldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI,and Fluorochem USA, etc. or synthesized according to sources known tothose skilled in the art (see, for example, Advanced Organic Chemistry:Reactions, Mechanisms, and Structure, 5th edition (Wiley, December2000)) or prepared as described in this invention.

Reaction Scheme I illustrates an exemplary method for preparing anintermediate useful for preparation of compounds of structure (I), whereR¹, L², L³ and M are as defined above, R² and R³ are as defined above orare protected variants thereof and L is an optional linker. Referring toReaction Scheme 1, compounds of structure a can be purchased or preparedby methods well-known to those of ordinary skill in the art. Reaction ofa with M-X, where x is a halogen such as bromo, under Suzuki couplingconditions known in the art results in compounds of structure b.Compounds of structure b can be used for preparation of compounds ofstructure (I) as described below.

Reaction Scheme II illustrates an alternative method for preparation ofintermediates useful for preparation of compounds of structure (I).Referring to reaction Scheme II, where R¹, L¹, L², L³, G and M are asdefined above, and R² and R³ are as defined above, or are protectedvariants thereof, a compound of structure c, which can be purchased orprepared by well-known techniques, is reacted with M-G′ to yieldcompounds of structure d. Here, G and G′ represent functional groupshaving complementary reactivity (i.e., functional groups which react toform a covalent bond). G′ may be pendant to M or a part of thestructural backbone of M. G and G′ may be any number of functionalgroups described herein, such as alkyne and azide, respectively, amineand activated ester, respectively or amine and isothiocyanate,respectively, and the like.

The compound of structure (I) may be prepared from one of structures bor d by reaction under well-known automated DNA synthesis conditionswith a phosphoramidite compound having the following structure (e):

wherein A is as defined herein and each L is independently an optionallinker.

DNA synthesis methods are well-known in the art. Briefly, two alcoholgroups, for example R² and R³ in intermediates b or d above, arefunctionalized with a dimethoxytrityl (DMT) group and a2-cyanoethyl-N,N-diisopropylamino phosphoramidite group, respectively.The phosphoramidite group is coupled to an alcohol group, typically inthe presence of an activator such as tetrazole, followed by oxidation ofthe phosphorous atom with iodine. The dimethoxytrityl group can beremoved with acid (e.g., chloroacetic acid) to expose the free alcohol,which can be reacted with a phosphoramidite group. The 2-cyanoethylgroup can be removed after oligomerization by treatment with aqueousammonia.

Preparation of the phosphoramidites used in the oligomerization methodsis also well-known in the art. For example, a primary alcohol (e.g., R³)can be protected as a DMT group by reaction with DMT-C₁. A secondaryalcohol (e.g., R²) is then functionalized as a phosphoramidite byreaction with an appropriate reagent such as 2-cyanoethylN,N-dissopropylchlorophosphoramidite. Methods for preparation ofphosphoramidites and their oligomerization are well-known in the art anddescribed in more detail in the examples.

Compounds of structure (I) are prepared by oligomerization ofintermediates b or d and e according to the well-known phophoramiditechemistry described above. The desired number of m and n repeating unitsis incorporated into the molecule by repeating the phosphoramiditecoupling the desired number of times. It will be appreciated thatcompounds of structure (II) as, described below, can be prepared byanalogous methods.

In various other embodiments, compounds useful for preparation of thecompound of structure (I) are provided. The compounds can be prepared asdescribed above in monomer, dimer and/or oligomeric form and then the Mmoiety covalently attached to the compound via any number of syntheticmethodologies (e.g., the “click” reactions described above) to form acompound of structure (I). Accordingly, in various embodiments acompound is provided having the following structure (II):

or a stereoisomer, salt or tautomer thereof, wherein:

G is, at each occurrence, independently a moiety comprising a reactivegroup, or protected analogue thereof, capable of forming a covalent bondwith a complementary reactive group;

L^(1a), L² and L³ are, at each occurrence, independently an optionalalkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene,heteroalkynylene or heteroatomic linker;

L⁴ is, at each occurrence, independently a heteroalkylene,heteroalkenylene or heteroalkynylene linker of greater than three atomsin length, wherein the heteroatoms in the heteroalkylene,heteroalkenylene and heteroalkynylene linker are selected from O, N andS;

R¹ is, at each occurrence, independently H, alkyl or alkoxy;

R² and R³ are each independently H, OH, SH, alkyl, alkoxy, alkylether,heteroalkyl, —OP(═R_(a))(R_(b))R_(c), Q or L′;

R⁴ is, at each occurrence, independently OH, SH, O⁻, S⁻, OR_(d) orSR_(d);

R⁵ is, at each occurrence, independently oxo, thioxo or absent;

R_(a) is O or S;

R_(b) is OH, SH, O⁻, S⁻, OR_(d) or SR_(d);

R_(c) is OH, SH, O⁻, S, OR_(a), OL′, SR_(a), alkyl, alkoxy, alkylether,alkoxyalkylether, phosphate, thiophosphate, phosphoalkyl,thiophosphoalkyl, phosphoalkylether or thiophosphoalkylether;

R_(d) is a counter ion;

Q is, at each occurrence, independently a moiety comprising a reactivegroup, or protected analogue thereof, capable of forming a covalent bondwith an analyte molecule, targeting moiety, a solid support or acomplementary reactive group Q′;

L′ is, at each occurrence, independently a linker comprising a covalentbond to Q, a linker comprising a covalent bond to a targeting moiety, alinker comprising a covalent bond to an analyte molecule, a linkercomprising a covalent bond to a solid support, a linker comprising acovalent bond to a solid support residue, a linker comprising a covalentbond to a nucleoside or a linker comprising a covalent bond to a furthercompound of structure (II);

m is, at each occurrence, independently an integer of zero or greater,provided that at least one occurrence of m is an integer of one orgreater; and

n is an integer of one or greater.

In other embodiments of structure (II):

G is, at each occurrence, independently a moiety comprising a reactivegroup capable of forming a covalent bond with a complementary reactivegroup;

L^(1a), L² and L³ are, at each occurrence, independently an optionalalkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene,heteroalkynylene or heteroatomic linker;

L⁴ is, at each occurrence, independently a heteroalkylene,heteroalkenylene or heteroalkynylene linker of greater than three atomsin length, wherein the heteroatoms in the heteroalkylene,heteroalkenylene and heteroalkynylene linker are selected from O, N andS;

R¹ is, at each occurrence, independently H, alkyl or alkoxy;

R² and R³ are each independently H, OH, SH, alkyl, alkoxy, alkylether,—OP(═R_(a))(R_(b))R_(c), Q, a linker comprising a covalent bond to Q, alinker comprising a covalent bond to an analyte molecule, a linkercomprising a covalent bond to a solid support or a linker comprising acovalent bond to a further compound of structure (II), wherein: R_(a) isO or S; R_(b) is OH, SH, O⁻, S⁻, OR_(d) or SR_(d); R_(c) is OH, SH, O⁻,S⁻, OR_(d), SR_(d), alkyl, alkoxy, alkylether, alkoxyalkylether,phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl,phosphoalkylether or thiophosphoalkylether; and R_(d) is a counter ion;

R⁴ is, at each occurrence, independently OH, SH, O⁻, S⁻, OR_(a) orSR_(d);

R⁵ is, at each occurrence, independently oxo, thioxo or absent;

Q is, at each occurrence, independently a moiety comprising a reactivegroup capable of forming a covalent bond with an analyte molecule, asolid support or a complementary reactive group Q′;

m is, at each occurrence, independently an integer of zero or greater,provided that at least one occurrence of m is an integer of one orgreater; and

n is an integer of one or greater.

The G moiety in the compound of structure (II) can be selected from anymoiety comprising a group having the appropriate reactivity group forforming a covalent bond with a complementary group on an M moiety. Inexemplary embodiments, the G moiety can be selected from any of the Qmoieties described herein, including those specific examples provided inTable 1. In some embodiments, G comprises, at each occurrence,independently a moiety suitable for reactions including: the coppercatalyzed reaction of an azide and alkyne to form a triazole (Huisgen 1,3-dipolar cycloaddition), reaction of a diene and dienophile(Diels-Alder), strain-promoted alkyne-nitrone cycloaddition, reaction ofa strained alkene with an azide, tetrazine or tetrazole, alkene andazide [3+2] cycloaddition, alkene and tetrazine inverse-demandDiels-Alder, alkene and tetrazole photoreaction and various displacementreactions, such as displacement of a leaving group by nucleophilicattack on an electrophilic atom.

In some embodiments, G is, at each occurrence, independently a moietycomprising an aldehyde, oxime, hydrazone, alkyne, amine, azide,acylazide, acylhalide, nitrile, nitrone, sulfhydryl, disulfide, sulfonylhalide, isothiocyanate, imidoester, activated ester, ketone,α,β-unsaturated carbonyl, alkene, maleimide, a-haloimide, epoxide,aziridine, tetrazine, tetrazole, phosphine, biotin or thiiranefunctional group.

In other embodiments, G comprises, at each occurrence, independently analkyne or an azide group. In other embodiments, G comprises, at eachoccurrence, independently an amino, isothiocyanate or activated estergroup. In different embodiments, G comprises, at each occurrence,independently a reactive group capable of forming a functional groupcomprising an alkene, ester, amide, thioester, disulfide, carbocyclic,heterocyclic or heteroaryl group, upon reaction with the complementaryreactive group. For example, in some embodiment the heteroaryl istriazolyl.

In various other embodiments of the compound of structure (II), L² andL³ are, at each occurrence, independently C₁-C₆ alkylene, C₂-C₆alkenylene or C₂-C₆ alkynylene.

In other embodiments, the compound has the following structure (IIA):

wherein:

x¹, x², x³ and x⁴ are, at each occurrence, independently an integer from0 to 6.

In other embodiments of structure (II), each L^(1a) is absent. In otherembodiments, each L^(1a) is present, for example L^(1a) is, at eachoccurrence, independently heteroalkylene. In certain embodiments, L^(1a)has the following structure:

In other of any of the foregoing embodiments of compound (II), G is, ateach occurrence, independently

In various embodiments of the compound of structure (IIA), at least oneoccurrence of x¹, x², x³ or x⁴ is 1. In other embodiments, x¹, x², x³and x⁴ are each 1 at each occurrence. In other embodiments, x¹ and x³are each 0 at each occurrence. In some embodiments, x² and x⁴ are each 1at each occurrence. In still other embodiments, x¹ and x³ are each 0 ateach occurrence, and x² and x⁴ are each 1 at each occurrence.

In some other embodiments of the compound of structure (II) or (IIA), L⁴is at each occurrence, independently a heteroalkylene linker. In othermore specific embodiments, L⁴ is at each occurrence, independently analkylene oxide linker. For example, in some embodiments L⁴ ispolyethylene oxide, and the compound has the following structure (IB):

wherein z is an integer from 2 to 100, for example an integer from 3 to6.

In other embodiments, R⁴ is, at each occurrence, independently OH, O⁻ orOR_(d), and in different embodiments R⁵ is, at each occurrence, oxo.

In some different embodiments of any of the foregoing compounds ofstructure (II) or (IIa), R¹ is H.

In other various embodiments of the compounds of structure (II), R² andR³ are each independently OH or —OP(═R_(a))(R_(b))R_(c). In somedifferent embodiments, R² or R³ is OH or —OP(═R_(a))(R_(b))R_(c), andthe other of R² or R³ is Q or a linker comprising a covalent bond to Q.

In still more different embodiments of any of the foregoing compounds ofstructure (II), R² and R³ are each independently—OP(═R_(a))(R_(b))R_(c). In some of these embodiments, R_(c) is OL′.

In other embodiments of structure (II), R² and R³ are each independently—OP(═R_(a))(R_(b))OL′, and L′ is a heteroalkylene linker to: Q, atargeting moiety, an analyte (e.g., analyte molecule), a solid support,a solid support residue, a nucleoside or a further compound of structure(II).

The linker L′ can be any linker suitable for attaching Q, a targetingmoiety, an analyte (e.g., analyte molecule), a solid support, a solidsupport residue, a nucleoside or a further compound of structure (II) tothe compound of structure (II). Advantageously certain embodimentsinclude use of L′ moieties selected to increase or optimize watersolubility of the compound. In some certain embodiments, L′ comprises analkylene oxide or phosphodiester moiety, or combinations thereof.

In certain embodiments, L′ has the following structure:

wherein:

m″ and n″ are independently an integer from 1 to 10;

R^(e) is H, an electron pair or a counter ion;

L″ is R^(e) or a direct bond or linkage to: Q, a targeting moiety, ananalyte (e.g., analyte molecule), a solid support, a solid supportresidue, a nucleoside or a further compound of structure (II).

In certain of the foregoing embodiments, the targeting moiety is anantibody or cell surface receptor antagonist.

In other more specific embodiments f any of the foregoing compounds ofstructure (II), R² or R³ has one of the following structures:

Certain embodiments of compounds of structure (II) can be preparedaccording to solid-phase synthetic methods analogous to those known inthe art for preparation of oligonucleotides. Accordingly, in someembodiments, L′ is a linkage to a solid support, a solid support residueor a nucleoside. Solid supports comprising an activated deoxythymidine(dT) group are readily available, and in some embodiments can beemployed as starting material for preparation of compounds of structure(II). Accordingly, in some embodiments R² or R³ has the followingstructure:

In still other embodiments of compounds of structure (II), Q is, at eachoccurrence, independently a moiety comprising a reactive group capableof forming a covalent bond with an analyte molecule or a solid support.In other embodiments, Q is, at each occurrence, independently a moietycomprising a reactive group capable of forming a covalent bond with acomplementary reactive group Q′. For example, in some embodiments, Q′ ispresent on a further compound of structure (II) (e.g., in the R² or R³position), and Q and Q′ comprise complementary reactive groups such thatreaction of the compound of structure (II) and the further compound ofstructure (II) results in covalently bound dimer of the compound ofstructure (II). Multimer compounds of structure (II) can also beprepared in an analogous manner and are included within the scope ofembodiments of the invention.

The type of Q group and connectivity of the Q group to the remainder ofthe compound of structure (II) is not limited, provided that Q comprisesa moiety having appropriate reactivity for forming the desired bond.

In certain embodiments of compounds of structure (II), the Q is a moietywhich is not susceptible to hydrolysis under aqueous conditions, but issufficiently reactive to form a bond with a corresponding group on ananalyte molecule or solid support (e.g., an amine, azide or alkyne).

Certain embodiments of compounds of structure (II) comprises Q groupscommonly employed in the field of bioconjugation. For example in someembodiments, Q comprises a nucleophilic reactive group, an electrophilicreactive group or a cycloaddition reactive group. In some more specificembodiments, Q comprises a sulfhydryl, disulfide, activated ester,isothiocyanate, azide, alkyne, alkene, diene, dienophile, acid halide,sulfonyl halide, phosphine, c-haloamide, biotin, amino or maleimidefunctional group. In some embodiments, the activated ester is anN-succinimide ester, imidoester or polyflourophenyl ester. In otherembodiments, the alkyne is an alkyl azide or acyl azide.

Exemplary Q moieties for compounds of structure (II) are provided inTable I above.

As with compounds of structure (I), in some embodiments of compounds ofstructure (II), wherein Q is SH, the SH moiety will tend to formdisulfide bonds with another sulfhydryl group on another compound ofstructure (II). Accordingly, some embodiments include compounds ofstructure (II), which are in the form of disulfide dimers, the disulfidebond being derived from SH Q groups.

In some other embodiments of compounds of structure (II), one of R² orR³ is OH or —OP(═R_(a))(R_(b))R_(c), and the other of R² or R³ is alinker comprising a covalent bond to an analyte molecule, a linkercomprising a covalent bond to a targeting moiety or a linker comprisinga covalent bond to a solid support. For example, in some embodiments theanalyte molecule is a nucleic acid, amino acid or a polymer thereof. Inother embodiments, the analyte molecule is an enzyme, receptor, receptorligand, antibody, glycoprotein, aptamer or prion. In some embodiments,the targeting moiety is an antibody or cell surface receptor antagonist.In still different embodiments, the solid support is a polymeric bead ornonpolymeric bead.

In other embodiments of compounds of structure (II), m is, at eachoccurrence, independently an integer from 1 to 10. For example, in someembodiments m is, at each occurrence, independently an integer from 1 to5, such as 1, 2, 3, 4 or 5. In some embodiments, m is 1. In someembodiments, m is 2. In some embodiments, m is 3. In some embodiments, mis 4. In some embodiments, m is 5.

In yet different embodiments of compounds of structure (II), n is aninteger from 1 to 100. For example, in some embodiments n is an integerfrom 1 to 10. In some embodiments, n is 1. In some embodiments, n is 2.In some embodiments, n is 3. In some embodiments, n is 4. In someembodiments, n is 5. In some embodiments, n is 6. In some embodiments, nis 7. In some embodiments, n is 8. In some embodiments, n is 9. In someembodiments, n is 10.

In other different embodiments, the compound of structure (II) isselected from Table 3.

TABLE 3 Exemplary Compounds of Structure (II) No. Structure I-1

II-2

II-3

II-4

II-5

II-6

II-7

II-8

II-9

II-10

II-11

II-12

II-13

II-14

II-15

II-16

II-17

II-18

II-19

II-20

II-21

II-22

II-23

II-24

II-25

II-26

II-27

II-28

II-29

II-30

In various embodiments, G in the compounds of Table 3 is alkynyl, suchas ethynyl. In other embodiments, G in the compounds of Table 3 is anazide. In other embodiments, G in the compounds of Table 3 is amino(NH₂). In other embodiments, G in the compounds of Table 3 is anisothiocyanate. In other embodiments, G in the compounds of Table 3 isan activated ester, such as an ester of N-hydroxysuccinimide.

The compounds of structure (II) can be used in various methods, forexample in embodiments is provided a method for labeling an analyte,such as an analyte molecule, or targeting moiety, the method comprising:

-   -   (a) admixing any of the described compounds of structure (II),        wherein R² or R³ is Q or a linker comprising a covalent bond to        Q, with the analyte molecule;    -   (b) forming a conjugate of the compound and the analyte or        targeting moiety; and    -   (c) reacting the conjugate with a compound of formula        M-L^(1b)-G′, thereby forming at least one covalent bond by        reaction of at least one G and at least one G′,

wherein:

M is a moiety comprising two or more carbon-carbon double bonds and atleast one degree of conjugation;

L^(1b) is an optional alkylene, heteroalkylene or heteroatomic linker;and

G′ is a reactive group complementary to G.

A different embodiment is a method for labeling an analyte, such as ananalyte molecule or targeting moiety, the method comprising:

-   -   (a) admixing any of the compounds of structure (II) disclosed        herein, wherein R² or R³ is Q or a linker comprising a covalent        bond to Q, with a compound of formula M-L^(1b)-G′, thereby        forming at least one covalent bond by reaction of G and G′; and    -   (b) reacting the product of step (A) with the analyte or        targeting moiety, thereby forming a conjugate of the product of        step (A) and the analyte molecule,

wherein:

M is a moiety comprising two or more carbon-carbon double bonds and atleast one degree of conjugation;

L^(1b) is an optional alkylene, heteroalkylene or heteroatomic linker;and

G′ is a reactive group complementary to G.

Further, as noted above, the compounds of structure (II) are useful forpreparation of compounds of structure (I). Accordingly, in oneembodiment is provided a method for preparing a compound of structure(I), the method comprising admixing a compound of structure (II) with acompound of formula M-L^(1b)-G′, thereby forming at least one covalentbond by reaction of G and G′, wherein:

M is a moiety comprising two or more carbon-carbon double bonds and atleast one degree of conjugation;

L^(1b) is an optional alkylene, heteroalkylene or heteroatomic linker;and

G′ is a reactive group complementary to G.

The following examples are provided for purposes of illustration, notlimitation.

EXAMPLES General Methods

Mass spectral analysis was performed on a Waters/Micromass Quattro microMS/MS system (in MS only mode) using MassLynx 4.1 acquisition software.Mobile phase used for LC/MS on dyes was 100 mM1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), 8.6 mM triethylamine (TEA), pH8. Phosphoramidites and precursor molecules were also analyzed using aWaters Acquity UHPLC system with a 2.1 mm×50 mm Acquity BEH-C18 columnheld at 45° C., employing an acetonitrile/water mobile phase gradient.Molecular weights for monomer intermediates were obtained usingtropylium cation infusion enhanced ionization on a Waters/MicromassQuattro micro MS/MS system (in MS only mode). Excitation and emissionprofiles experiments were recorded on a Cary Eclipse spectra photometer.

All reactions were carried out in oven dried glassware under a nitrogenatmosphere unless otherwise stated. Commercially available DNA synthesisreagents were purchased from Glen Research (Sterling, Va.). Anhydrouspyridine, toluene, dichloromethane, diisopropylethyl amine,triethylamine, acetic acid, pyridine, and THF were purchased fromAldrich. All other chemicals were purchase from Aldrich or TCI and wereused as is with no additional purification.

Example 1 Synthesis of Dyes with Ethylene Glycol Spacer

Compounds with ethylene oxide linkers were prepared as followed:

The oligofluoroside constructs (i.e., compounds of structure (I)) weresynthesized on an Applied Biosystems 394 DNA/RNA synthesizer on 1 μmolscale and possessed a 3′-phosphate group or 3′-S₂—(CH₂)₆—OH group or anyof the other groups described herein. Synthesis was performed directlyon CPG beads or on Polystyrene solid support using standardphopshoporamadite chemistry. The oligofluorosides were synthesized inthe 3′ to 5′ direction using standard solid phase DNA methods, andcoupling employed standard β-cyanoethyl phosphoramidite chemistry.Fluoroside phosphoramidite and spacers (e.g., hexaethyloxy-glycolphosphoramidite, triethyloxy-glycol phosphoramidite, polyethylene glycolphosphoramidite) and linker (e.g., 5′-amino-Modifier Phosphoramidite andthiol-Modifiers S2 Phosphoramidite) were dissolved in acetonitrile tomake 0.1 M solutions, and were added in successive order using thefollowing synthesis cycle: 1) removal of the 5′-dimethoxytritylprotecting group with dichloroacetic acid in dichloromethane, 2)coupling of the next phosphoramidite with activator reagent inacetonitrile, 3) oxidation of P(III) to form stable P(v) withiodine/pyridine/water, and 4) capping of any unreacted 5′-hydroxylgroups with acetic anhydride/1-methylimidizole/acetonitrile. Thesynthesis cycle was repeated until the full length oligofluorosideconstruct was assembled. At the end of the chain assembly, themonomethoxytrityl (MMT) group or dimethoxytrityl (DMT) group was removedwith dichloroacetic acid in dichloromethane.

The compounds were provided on controlled-pore glass (CPG) support at0.2 umol scale in a labeled Eppendorf tube. 400 μL of 20-30% NH₄OH wasadded and mixed gently. Open tubes were placed at 55° C. for ˜5 minutesor until excess gases had been liberated, and then were closed tightlyand incubated for 2 hrs (+/−15 min.). Tubes were removed from the heatblock and allowed to reach room temperature, followed by centrifugationat 13,400 RPM for 30 seconds to consolidate the supernatant and solids.Supernatant was carefully removed and placed into a labeled tube, andthen 150 μL acetonitrile was added to wash the support. After the washwas added to the tubes they were placed into a CentriVap apparatus at40° C. until dried.

The products were characterized by ESI-MS (see Table 2), UV-absorbance,and fluorescence spectroscopy.

Example 2 Spectral Testing of Compounds

Dried compounds were reconstituted in 150 μL of 0.1M Na₂CO₃ buffer tomake a ˜1 mM stock. The concentrated stock was diluted 50× in 0.1×PBSand analyzed on a NanoDrop UV spectrometer to get an absorbance reading.Absorbance readings were used along with the extinction coefficient(75,000 M⁻¹ cm⁻¹ for each FAM unit) and Beer's Law to determine anactual concentration of the stock.

From the calculated stock concentrations, ˜4 mL of a 5 μM solution wasmade in 0.1M Na₂CO₃ (pH 9) and analyzed in a 1×1 cm quartz cuvette on aCary 60 UV spectrometer, using a spectral range of 300 nm to 700 nm, togauge overall absorbance relative to the group. From these 5 μMsolutions, a second dilution was made at either 50 nM or 25 nM (also in0.1M Na₂CO₃, pH 9) for spectral analysis on a Cary Eclipse Fluorimeter.Excitation was set at 494 nm and emission spectra were collected from499 to 700 nm.

FIG. 1 and FIG. 2 provide the UV absorbance of representative compoundsof structure (I) and a comparative compound (“Compound A.”) As seen inFIGS. 1 and 2, the UV extinction coefficient of representative compoundsof structure (I) comprising two fluorescein moieties is approximatelytwice that of compound A.

The fluorescence emission spectra of representative compounds ofstructure (I) were also determined and compared to the emission spectrumof compound A. As demonstrated by the data in FIGS. 3 and 4, thefluorescence emission of representative compounds of structure (I) ishigher than compound A, and the emission increases as the number oftriethylene glycol or hexaethylene glycol units increases. While notwishing to be bound by theory, it is believed this unexpected increasein fluorescence emission is related to a decrease in internal quenchingassociated with the spatial distance provided by L⁴.

Compounds I-10 and I-11 were tested to determine the effect of thenumber of M moieties on the UV absorbance and fluorescence emission ofthe compounds. FIG. 5 provides data comparing UV absorbance of compoundsI-10 and I-11 to a comparative compound having a single M moiety(“Compound B”) at 5 m. At 5 uM, Compound B, which contains a single FAMunit absorbed at 0.43 AU, while compound I-10 (3 FAM units) absorbed at1.17 AU and compound I-11 (5 FAM units) absorbed at 2.00 AU.

Fluorescence emission spectra for compounds I-10, I-11 and B at 25 nMare presented in FIG. 6. Rather than quenching (as more closely-spacedFAM units would do), compounds I-10 and I-11 showed emission responsesthat were increased by 2.5× and 4.3×, respectively, compared to thevalue of Compound B.

Example 3 Comparative Fluorescence Emission Response

Compounds “HEG,” “TEG,” “C2,” “C3,” “C4” and “C6,” wherein R² and R³ areas defined for compound I-3 and m varied from 1 to 9, were prepared andtheir fluorescence emission spectra determined. Results are presented inFIG. 7. The data show that compounds according to embodiments of thepresent invention (i.e., HEG and TEG) have increased fluorescenceemission with fewer repeating spacer moieties (i.e., lower values of m)relative to other dye compounds.

FIG. 8 provides data comparing fluorescence emission for the “HEG”compound, wherein m is 1, 2 or 3, relative to Compound A (50 nM, pH=9).The data show an increase in fluorescence emission for HEG relative toCompound A when m is greater than 2.

Example 4 Preparation of Representative Compounds

Compounds I-29, I-32 and representative analogues were prepared andtested to determine whether compounds wherein L⁴ is a long linker(˜1,000 dalton PEG) have similar properties to compounds with shorter L⁴linkers, but with multiple repeats (i.e., m is greater than 1). FIG. 9provides UV absorbance data for compound I-60, compound I-46 andCompound B. The data show that compounds with long L⁴ linkers have UVabsorbance similar to those of compounds with multiple repeats ofshorter linkers, and both compounds have increased absorbance relativeto the control Compound B.

Example 5 Preparation of 99-Mer Dye

Compound I-42, having 33 fluorescein moieties was prepared usingstandard solid-phase oligonucleotide techniques as described herein.I-42 (represented by “A” in the below scheme) was trimerized asillustrated and described below to form a 99-mer dye.

In a 200 μL polypropylene tube was placed sodium phosphate buffer (3.5μL, 100 mM, pH=6.5) and a solution of I-42 bis-disulfide (5.5 μL, 0.18mM in water). To this was added a solution tris(2-carboxyethyl)phosphine(TCEP, 1.0 μL, 10 mM in water). The tube was capped, vortexed andallowed to incubate at room temperature for 2 h. The mixture wasdesalted through micro Zeba Spin desalting columns (Pierce, Cat #89877).The desalted solution was treated with sodium phosphate buffer (2.0 μL,500 mM, pH=7.2) and a DMSO solution of bismaleimidoethane (BMOE, 1.0 μL,0.25 mM) and incubated overnight at room temperature. The reactionmixture was diluted with water (100 μL) and analyzed by PAGE (FIG. 10,Invitrogen EC6875, 10% TBE-Urea gel, 180V constant, electrophoresishalted with resolution of highest MW species completed, visualized by UVillumination (365 nm)).

Other oligomer dyes having any desired number of dye moieties areprepared in an analogous manner.

Example 6 General Flow Cytometry Methods

Unless otherwise noted, the following general procedures were used inthroughout the following Examples.

Lysis of Whole Blood: Buffered Ammonium Chloride Method.

For staining of live cells, ethylenediaminetetraacetate (EDTA)anticoagulated normal human blood is bulk lysed with Ammonium Chloridesolution (ACK), 15 mL blood to 35 mL lyse for 15 min at room temperature(RT). The cells were washed twice with 50% Hank's Balanced Salt Solution(HBSS) and 50% 1% Fetal Bovine Serum (FBS) 1× Dulbecco'sPhosphate-Buffered Saline (DPBS) with 0.02% sodium azide. The cells werethen re-suspended to 100 μL/test/0.1-1×10e6 in donor plasma. Cells inplasma were added to pre-diluted antibodies for Vf of 100 μL 1% BovineSerum Albumin (BSA) and 1×DPBS with 0.02% sodium azide in polypropylene96 well HTS plates. After incubating for 45 min. at RT, the cells werewashed twice with 50% HBSS and 50%-1% FBS 1×DPBS with 0.02% sodiumazide.

Lyse/Fixation Method.

Blood was lysed with 1.0 mL RBC lysing solution (ammonium chloride),100-15 mL blood to 35 mL lyse for 15 min at RT. The cells were thenwashed twice with 50% HBSS and 50%-1% FBS 1×DPBS with 0.02% sodiumazide. Cells were then re-suspended to 100 μL/test/1×10e6 in donorplasma. Pre-diluted antibodies were added in 100 μL 1% BSA and 1×DPBSwith 0.02% sodium azide. 100 μL cells were added to 96 wellpolypropylene HTS plates (total 200 μL test size). After incubation for45 min. at RT the cells were washed twice with 50% HBSS and 50% 1% FBS1×DPBS with 0.02% sodium azide.

Preparation of Antibody Conjugates:

Antibody conjugates were prepared by reacting a compound of structure(I) comprising a Q moiety having the following structure:

with the desired antibody. The compound and antibody are thus conjugatedby reaction of an S on the antibody with the Q moiety to form thefollowing linking structure:

Antibody conjugates are indicated by the antibody name following by thecompound number. For example, UCHT1-I-45 indicates a conjugate formedbetween compound I-45 and the UCHT1 antibody. If a referenced compoundnumber does not include the above Q moiety in Table 2, it is understoodthat the Q moiety was installed and the conjugate prepared from theresulting compound having the Q moiety.

Dilution of Conjugates:

Antibodies were brought to RT. The antibody conjugates were diluted toconcentrations in a range of 0.1-540 nM (8.0 micrograms or less pertest) in a cell staining buffer (1×DPBS, 1% BSA, 0.02% sodium azide). Insome examples, serial dilutions for each sample started at 269 nMantibody in cell staining buffer, and the antibody dilutions were keptprotected from light until use. In other experiments, dilutions startedat 4.0 μg antibody/test size, with the test size ranging from 100-200μL. Titers were performed in two fold or four fold dilutions to generatebinding curves. In some cases, 8.0 or 2.0 μg/test size were used infirst well in a dilution series.

Flow Cytometry with Conjugate:

After physical characterization, the conjugates were tested for activityand functionality (antibody binding affinity and brightness of dye) andcompared to reference antibody staining. Then the quality of resolutionwas determined by reviewing the brightness in comparison toauto-fluorescent negative controls, and other non-specific binding usingthe flow cytometer. Extensive studies of the mouse IgG1,k isotypecontrol MOPC-21 conjugates were not included when testing I-45 becauseMOPC-21 non-specific binding was characterized during the testing ofUCHT1-Compound C and UCHT1-I-45 in earlier tests. The I-45 conjugateswere tested on Jurkat T cells, Ramos B cells, and a heterogeneouspopulation of leukocytes in human blood or peripheral blood mononuclearcells (PBMC), and using polystyrene goat-anti-mouse Ig coated beads.Whole blood screening was the most routine for testing UCHT1 I-45 andits analogues. Bridging studies were implemented as new constructs wereformed. Additional flow cytometry methods were used when testingconjugates (UCHT1-I-56, I-48, I-49, I-16, and I-21B) and compared toantibody conjugate references from Sony Biotechnology (UCHT1-FITC) andthe key bridging references previously characterized (e.g. UCHT1-I-45,UCHT1-I-49) in most studies.

Perform Free Dye Flow Cytometry:

After molecular and physical characterization, the dyes were also testedfor potential affinity to cells compared to a reference dye stain.Because dyes have the potential to also function as cellular probes andbind to cellular material, dyes can be generally screened against bloodat high concentrations (>100 nM-to-10,000 nM) to ascertain specificcharacteristics. Expected or unexpected off target binding was thenqualified by evaluating brightness and linearity upon dilution incomparison to auto-fluorescent negative controls, and other dye controlsusing the flow cytometer. Studies of Compound D (a Compound Cfree dye,but non-functionalized) was the positive control for bright off targetbinding of dyes and has been previously characterized when in conjugateform. The I-45 dyes were tested on heterogeneous population ofleukocytes in human blood when cells are treated with lysis and fixationsolution, and when the blood is aged, or when applied to PBMC monocytepopulations. Bridging studies ranking the affinity (Compound D, I-45,I-49, and I-16) were performed for dye lot comparisons while includingdyes from very early studies when characterizing Compound D.

Flow Cytometry Workflow:

Cells were cultured and observed for visual signs of metabolic stressfor dye screening or off target binding (data not shown), or freshhealthy cells were used for conjugate screening. Cells were countedperiodically to check cell density (1×10e5 and 1×10e6 viable cells/mL).Antibody conjugates were diluted (preferably in plate or tubes) beforeharvesting cells in stain buffer (DPBS, 0.1% BSA, 0.02% sodium azide).Cells with a viability range of 80-85% were used. The cells were washedtwice by centrifuging and washing cells with buffer to remove pHindicator, and to block cells with Ig and other proteins contained inFBS. The cell density was adjusted to test size in stain buffer. Thecells were plated, one test per well, or dyes (pre-diluted) were appliedto cells in plate. Then, the cells were incubated 45 min at 23° C. Thecells were washed twice by centrifuging and washing cells with washbuffer, then aspirating the plate. The cells were re-suspended inacquisition buffer. 5000 intact cells were acquired by flow cytometry.The fluorescence of the dyes was detected by 488 nM blue laser line byflow cytometry with peak emission (521 nM) detected using 525/50bandpass filter. At least 1500 intact cells, with target acquisitions of3000-5000 intact cells, were acquired by flow cytometry and analyzed toidentify viable cells present in the cell preparation.

Data Analysis Methods:

Descriptive Statistics.

The EC-800 software allows a user to collect numerous statistical datafor each sample acquisition. Mean or Median Fluorescence Intensity (MFI)in the FL1-A channel was used to measure the brightness of anantibody-dye reagent when it was being interrogated by flow cytometryand when noise was reviewed. Other statistics were evaluated todetermine dye characteristics and overall quality of the reagentsincluding median Signal-to-Noise and absolute fluorescence (median orGeomean).

Histograms.

The flow cytometry events were gated by size on forward versus sidescatter (cell volume versus cell granularity). Those cells were thengated by fluorescent emission at 515 nm for Mean Fluorescence Intensity(MFI). The data collected are presented as dual parameter histogramsplotted as number of events on the y-axis versus fluorescent intensity,which is represented on a log scale on the x-axis. The data may besummarized by affinity curves, or histograms of relative fluorescenceintensity.

Binding Curves.

MFI was chosen as it is the parameter that best measures the brightnessof an antibody-dye reagent when it is being interrogated by FCM, thiscan be expressed as the geometric mean, median, or mean, and representabsolute fluorescence measurements. For comparison, where the noise canbe highly characterized, a Signal-to-Noise ratio is reported as MFI,S/N. In Examples 7, 8, and 14, the MFI of the UCHT1-Compound Cconjugates versus concentration is shown to demonstrate binding curvesof the reagent.

Bi-Variate, Dual Parameter Histograms.

In some cases, the FCM events were not gated in order to reviewqualitative outputs, and data are expressed by cell granularity (SSC)versus dye fluorescence. This method allows for the overall evaluationof all populations recovered in whole blood.

Example 7 Evaluation of Dyes for Non-Specific and Off Target BindingUsing Necrotic and Apoptotic Populations of Heat Stressed Jurkat T Cells

Jurkat cells were cultured according to instructions provided byAmerican Type Culture Collection (ATCC), harvested live, heat stressed,washed 2-3×, and stained with conjugate antibodies. Staining wasperformed by applying cells to pre-diluted dyes and pre-dilutedconjugated antibodies, incubating, washing, and then acquiring by flowcytometry. The dead and necrotic cell population (˜10% of acquiredcells) was evaluated for fluorescence signal. The results are shown inFIG. 11. As shown in FIG. 11, fluorescence is observed to be higher in10× Compound D free dye compared to 10× I-47, 5× Compound E, 5× I-44, 3×Compound F and 3× I-43.

Example 8 Evaluation of Conjugates for Fluorescence Intensity:UCHT1-Compound G Vs. UCHT1-I-51

Viable Jurkat cells were cultured according to instructions provided byATCC and harvested, then recovered at ˜225 RCF for 6 minutes in atemperature controlled centrifuge set to 23° C. The supernatant wasremoved. Then the cells were washed twice in cell suspension buffer(calcium and magnesium free 1×DPBS, 1.0% FBS, 0.02% sodium azide, pH7.2). After the second wash, the cells were centrifuged, the supernatantwas removed, and the cells (˜5×10⁵ viable cells per sample) werere-suspended to test-size (final volume 100 μL). Cells were incubatedwith antibody dye conjugate solutions for 45 minutes at RT. After theincubation, samples were washed twice and then suspended in acquisitionbuffer.

Data were acquired and evaluated on the SONY EC-800 FCM, and plotted nMof antibody protein versus geometric mean of relative fluorescence, asshown in FIG. 12. As can be seen, MOPC-21-Compound Ghas non-specificbinding, yet both conjugates are 4-5× brighter than FITC reference. Thisexample also shows that MOPC21-I-51 shows reduced non-specific bindingcompared to UCHT1-Compound G conjugate (at these dye on label (DOL)levels).

Example 9 Evaluation of CD3 Expression (Specificity and Resolution) inHeterogeneous Cell Sample and Peripheral Whole Blood Cells

Whole blood was drawn from a normal donor into an EDTA stabilized sampletube for transport and short term storage. The blood was lysed with ACK,15 mL blood to 35 mL lyse for 15 min at RT. The cells were washed twicewith 50% Hanks Balanced Salt Solution (HBSS) and 50%-1% FBS 1×DPBS with0.02% sodium azide. The cells were re-suspended to 100 μL/test/1×10e6 indonor plasma. Antibodies were pre-diluted in 100 μL 1% BSA and 1×DPBSwith 0.02% sodium azide, and were added to 100 μL cells in polypropylene96 well HTS plates (total 200 μL test size). The cells were incubatedfor 45 min. at RT, washed twice with 50% HBSS and 50%-1% FBS 1×DPBS with0.02% sodium azide, and re-suspended in 1% FBS 1×DPBS with 0.02% sodiumazide.

FIG. 13 shows comparisons of I-51 conjugation and Compound G referenceantibody. In FIG. 13, the cell morphology (SSC-Lin) is shown in a dualparameter histogram with dye emission detected in the FL1-A channel.This shows the non-specific binding (NSB) of Compound G conjugate onheterogeneous population of cells, primarily neutrophils and monocyteswhile I-51 conjugates do not show NSB. The NSB of UCHT1-Compound G inlysed whole blood, effectively reduces the available antibody forbinding to CD3

FIG. 14 shows a comparison of UCHT1-I-51, UCHT1 BB515, and UCHT1-FITC.UCHT1-I-51 is 6× brighter than UCHT1-FITC.

Example 10 Expression Levels of CD3 Compared to a Molecules ofEquivalent Fluorochrome (MEF) Standard Curve

Using lysed whole blood, high antigen density CD3 expression wasvisualized by fluorescence intensity results and compared to 6-Peak (or8-peak) bead fluorescence outputs (Sony Biotech, Cat. No. AE700520) toestimate MEF Values. UCHT1-FITC, used as a reference, UCHT1-Compound G,and UCHT1-I-51, as well as UCHT1-BB515, used as an additional referenceconjugate, were compared in the same experiment using standards. 6 PeakBeads consisted of a mixture of 3.8 micron beads of 6 differentfluorescence intensities and were used to verify the linearity andsensitivity of the instrument and to estimate MEF when run in parallelin a given protocol.

FIG. 15 shows expression levels of CD3 compared to a MEF standard curve.As can be seen, I-51 is approximately 6× brighter than the reference,and Compound G is about 2× as bright. Concentration ranges shown are 133nM and below. Note that, in comparison, FIG. 13 shows that thenon-specific binding of UCHT1-Compound G in lysed whole bloodeffectively reduced the available antibody for binding to CD3.

Example 11 Comparison of UCHT1 I-16 Fractions to FITC and I-56Conjugates

Beads were pre-treated (vortexed and sonicated) and washed, and beadcounts calibrated to a 2×C dilution as determined in preliminaryexperiments to optimize acquisitions and target a linear saturationcurve. Beads were incubated with antibody conjugates, washed, and thenacquired by flow cytometry. BSA solution 0.1% in 1×DPBS was used forbead dilutions, washing, and acquisition. The antibodies werepre-diluted in 1% BSA Stain Buffer in 96 well polypropylene platesstarting at 4.0 μg in a 200 μL volume in first well, and then serialdiluted 100 μL in each subsequent well for at least 8 dilutions, (twofold). Thoroughly vortexed beads (at 2×C) were then added, and 100 μL ofbeads were added to 100 μL of antibody in each well. The beads wereincubated for 20 minutes at RT, washed, and acquired by flow cytometry.

The results are shown in FIG. 16. UCHT1-I-16 at DOL˜3.0 approachedtheoretical maximum. As shown in FIG. 17, a similar experiment wasperformed to highlight the affinity curve differences noted betweenUCTH1-I-16 and the bridging reference with a longer tether, UCHT1-I-56.

Example 12 UCHT1 I-51-Like Analogue, UCHT1 I-16, Compared to UCHT1 I-56(10×), and UCHT1 I-53 (6×)

Peripheral WBC were treated with lysis buffer, buffered ACK, for 20minutes at 25° C., while slow rocking, then centrifuged and the lysisbuffer removed. The cells were washed once with HBSS, pH 7.2, then1×HBSS containing 0.5% FBS, and 0.02% Sodium Azide, pH 7.2, andre-suspended in Staining Buffer (1×DPBS, 1% BSA, 0.02% Sodium Azide, Ph7.2). Cells were then applied to pre-diluted conjugated antibodies andincubated for 40 minutes at 23-25° C. protected from light.

FIG. 18 shows a comparison of the UCHT1-I-51-like analogue, UCHT1-I-16,with UCHT1-I-56 (10×), and UCHT1-I-53 (6×).

Example 13 UCHT1 I-51-Like Analogue, UCHT1 I-16, Compared to UCHT1 I-56(10×), and UCHT1 I-53 (6×)

Antibodies were evaluated for specific binding and fluorescenceresolution by flow cytometry. Jurkat cells were cultured according toinstructions provided by ATCC and harvested live. Staining was performedwhen cells were applied to pre-diluted conjugated antibodies, incubatedfor 20-40 minutes, washed, and then acquired by flow cytometry. TheUCHT1-I-51-like analogue, UCHT1-I-16, was compared with UCHT1-I-56(10×), and UCHT1-I-53 (6×). The results are shown in FIG. 19.

Example 14 Comparison of UCHT1 Conjugate Resolution by RegressionAnalysis

Cells were isolated from peripheral whole blood, and frozen in freezingbuffer. Cells were thawed, rested, treated with autologous plasma toblock FcR and to mimic a whole blood environment, washed two to threetimes, and then stained with conjugate much like when using whole blood.Beads were pre-calibrated to optimize acquisitions and target saturationin an antibody staining. Beads were incubated with antibody conjugates,washed, and then acquired by flow cytometry.

Regression analysis was performed on data produced when testingUCHT1-I-16 and UCHT1-I-49 to demonstrate equivalency betweenconjugations. The results are shown in FIG. 20.

Example 15 I-49 and I-16 Affinity Testing of Raw Dye in Whole Blood

The dye was screened using the stain, lyse, fix, and wash whole bloodmethod to evaluate background in three populations, monocytes,granulocytes, and lymphocytes, in an equivalency test in the presence ofexcess dye. Granulocytes present in the whole blood were chosen as themain target for analyses. Although lymphocytes and monocytes were alsostudied, data are not shown in regression graphs.

The raw dye was used in excess (first titration starting at 10,000 nM)without antibody conjugate being present in order to highlight, but alsoqualify, non-specific binding differences between the two nearlyidentical constructs. Peripheral WBC were treated with lysis buffer,buffered ACK, for 20 minutes at 25° C., while slow rocking, centrifuged,and the lysis buffer removed. A red cell lysis and fixation solution wasapplied to the dye and cells, and the cells were then washed once withHBSS, pH 7.2, then 1×HBSS containing 0.5% FBS, and 0.02% Sodium Azide,pH 7.2, and then re-suspended in Staining Buffer (1×DPBS, 1% BSA, 0.02%Sodium Azide, pH 7.2).

The Relative MFI data was concentration matched and compared byregression analyses to demonstrate level of agreement and or similaritybetween the raw I-45 and I-16. In this test, I-45 and I-16 are found tohave nearly equivalent levels of background. Using the same data fromdye screening, the I-45 analogues (I-49 and I-16) overlay each otherwhen examining the entire titration curve and the controls. I-45analogues fall in between MFIs of controls included for reference wereCompound D (10×), and two analogues Compound D (analogues i and ii)having longer alkylene spacer groups. I-49 has slightly higherbackground than I-16 and I-16 background is very similar to I-45. The10× fluorophore constructs are dimmer in non-specific binding than the6×, demonstrating effectiveness of backbone modulation to accommodateadditional fluorophores while reducing non-specific binding propertiesof the 10× molecule. The results are shown in FIGS. 21A-21C. FIG. 21Ashows correlations between I-16 and I-45 FIG. 21B shows titration curveoverlays and compared to references; and FIG. 21C shows examplequalitative data showing background FL and cell morphology comparingCompound D and I-45.

Example 16 Comparison of UCHT1-I-21B and UCHT1-I-16 in Blood Cells thathave been Fixed and Stored for 72 Hours

Whole blood was drawn from a normal donor into an EDTA stabilized sampletube for transport and short term storage. The blood was treated withlysing agents, either before or after staining with antibodies. Thecells were lysed with ACK, 15 mL blood to 35 mL lyse for 15 min at RT,and then washed twice with 50% HBSS and 50% 1% FBS 1×DPBS with 0.02%sodium azide. The cells were re-suspended to 100 μL/test/1×10e6 in donorplasma. Pre-diluted antibodies in 100 μL 1% BSA and 1×DPBS with 0.02%sodium azide were added to 100 μL cells, which were then added to 96well HTS polypropylene plates (total 200 μL test size). After incubatingthe cells for 45 min. at RT, the cells were washed twice with 50%HBSS+50% 1% FBS 1×DPBS with 0.02% sodium azide. The cells were thenre-suspended in 1% FBS 1×DPBS with 0.02% sodium azide. The cells werewashed once more, fixed in 2% paraformaldehyde at 200 μL/well, washedonce with 1×DPBS, stored for 72 hours 2-8° C., washed once more using1×DPBS, and then acquired using 0.1% BSA in 1×DPBS.

The resolution of the conjugates was compared to reference, UCHT1-FITC,and to theoretical brightness for a DOL of 3.0. The new constructUCHT1-I-21B best matches theoretical when DOL is 3.0 in this method, andis seven times brighter that UCHT1-FITC. As shown in FIG. 22, affinitycurves, as histograms, are shown with compound emission detected in theFL1-A channel.

An assessment of non-specific binding was completed by measuring thefluorescence of granulocytes. FIG. 23 shows comparisons of fluorescenceintensity of off target, non-specific binding of UCHT1-I-21B,UCHT1-I-16, and reference, UCHT1-FITC. All fractions and replicates ofUCHT1-I-21B show less background than other constructs and the FITCreference included in the test. Regression analysis was applied to thedata to review correlations and relative affinities, as shown in FIG.24, and it was determined that UCHT1-I-21B does not have a linearrelationship with UCHT1-I-16.

Example 17 UCHT1 I-21B Using the Jurkat Cell Model and a Simple TwoPoint Titer

Similar to Example 16 a test of UCHT1 I-21B was performed in a simpletwo point titer of 2.0 and 0.125 micrograms per test of antibody. Jurkatcells were cultured according to instructions provided by ATCC,harvested live or heat stressed, washed 2-3×, and then stained withconjugate antibodies. Staining was performed when cells were applied topre-diluted conjugated antibodies, incubated, washed, and then acquiredby flow cytometry.

As shown in FIG. 25, UCHT1-I-21B demonstrates higher affinity at lowconcentration compared to UCHT1-I-51, as expected. The actual signal tonoise exceeds theoretical at a sub saturation C of 0.125 micrograms pertest and out performs UCHT1-I-51.

Example 18 Comparison of UCHT1 Compound G and UCHT1 I-51 in a PlasmaInterference Study Using PBMC

PBMC and autologous plasma were previously isolated from peripheralwhole blood, and then frozen in freezing media. Cells were thawed,briefly rested, washed two or three times, and then stained withconjugate antibodies as if freshly isolated from whole blood, withautologous plasma, or HBSS present during antibody staining.

UCHT1-Compound G interacted with monocytes to form fluorescent events inthe presence of activated and deactivated donor plasma, and additions of2.5% glycine. FIG. 26A shows data resulting from the addition of 0%glycine, and FIG. 26B shows data resulting from the addition of 2.5%glycine. The Zwitterion amino acid glycine plays a role in immunoassaysas surrogate affinity for, amide binding with, or blocking by, naturalpoly-amines, thus exaggerating or blocking the effects of other reagentsin the staining system of live cells. Plasma re-introduced to PBMC iseither (1) activating, with compliment, platelets present at normallevels, or (2) deactivated (both plasma compliment and other factors) byheat, filtration, and centrifugation to remove most platelets. Thedeactivated plasma functions more as a blocking agent, while theactivating plasma is expected to have high interference.

The study mimics a range of effects possible in whole blood when bloodis mobilized, plasma is present, residual, diluted, or washed away.Compound C and I-45 show distinct differences in behavior as expected,supporting a decrease in background binding and distinct improvement inactivity by structural modification to I-45. Generally, it is observedthat UCHT1-I-51 background is limited in comparison to Compound G, whileglycine when present slightly enhances the background staining of I-51and suppresses the background of UCHT1-Compound G, particularly in thedeactivated plasma and neat control. Overall, the UCHT1-Compound G hashigher monocyte background.

Example 19 Preparation of Phosphoramidites and Compounds

Exemplary compounds were prepared using standard solid-phaseoligonucleotide synthesis protocols and a fluorescein-containingphosphoramidite having the following structure:

which was purchased from ChemGenes (Cat. # CLP-9780).

Exemplary linkers (L⁴) were included in the compounds by coupling with aphosphoramidite having the following structure:

which is also commercially available.

Other exemplary compounds were prepared using a phosphoramidite preparedaccording to the following scheme:

Final Deprotection produces the desired F″ moiety. Other commerciallyavailable phosphoramidite reagents were employed as appropriate toinstall the various portions of the compounds. Q moieties having thefollowing structure:

were installed by reaction of:

with a free sulfhydryl. Other Q moieties are installed in an analogousmanner according to knowledge of one of ordinary skill in the art.

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification areincorporated herein by reference, in their entirety to the extent notinconsistent with the present description.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1-129. (canceled)
 130. A compound having the following structure (IB):

or a stereoisomer or salt thereof wherein: M is, at each occurrence,independently a fluorescent dye; R¹ is, at each occurrence, H; R² is alinker comprising a covalent bond to a targeting moiety; R³ is OH, SH,—OP(═R_(a))(R_(b))R_(c) or L′; R⁴ is, at each occurrence, independentlyOH, SH, O⁻, S⁻, OR_(d) or SR_(d); R⁵ is, at each occurrence,independently oxo or thioxo; R_(a) is O or S; R_(b) is OH, SH, O⁻, S⁻,OR_(d) or SR_(d); R_(c) is OH, SH, O⁻, S⁻, OR_(d), OL′ or SR_(d); R_(d)is a cation; L′ is a linker a linker comprising a covalent bond to asolid support, a linker comprising a covalent bond to a solid supportresidue or a linker comprising a covalent bond to a nucleoside; L¹ is ateach occurrence, independently an alkylene or heteroalkylene linker; mis, at each occurrence, independently an integer of one or greater; n isan integer of one or greater; x¹, x², x³ and x⁴ are, at each occurrence,independently an integer from 0 to 6; and z is an integer from 2 to 100.131. The compound of claim 130, wherein the targeting moiety selectivelybinds to a biomolecule.
 132. The compound of claim 130, wherein thebiomolecule is an enzyme, receptor, receptor ligand, antibody, bacteria,virus, blood cell or tissue cell.
 133. The compound of claim 130,wherein x¹, x², x³ and x⁴ are each 1 at each occurrence.
 134. Thecompound of claim 130, wherein R⁴ is, at each occurrence, independentlyOH, O⁻ or OR_(d), and R⁵ is, at each occurrence, oxo.
 135. The compoundof claim 130, wherein R³ is OH.
 136. The compound of claim 130, whereinR³ is —OP(═R_(a))(R_(b))R_(c).
 137. The compound of claim 136, whereinR_(c) is OL′.
 138. The compound of claim 137, wherein L′ has thefollowing structure:

wherein: m″ and n″ are independently an integer from 1 to 10; R^(e) isH, an electron pair or a counter ion; and L″ is R^(e) or a direct bondor linkage to a solid support, a solid support residue or a nucleoside.139. The compound of claim 130, wherein R³ has the following structure:


140. The compound of claim 130, wherein R³ has the following structure:


141. The compound of claim 130, wherein L¹, at each occurrence,independently has one of the following structures:


142. The compound of claim 130, wherein m is, at each occurrence,independently an integer from 1 to
 10. 143. The compound of claim 130,wherein m is, at each occurrence, independently 3, 4, 5 or 6, and z isan integer from 3 to
 6. 144. The compound of claim 130, wherein n is aninteger from 1 to
 10. 145. The compound of claim 130, wherein z is aninteger from 3 to
 10. 146. The compound of claim 130, wherein z is aninteger from 20 to
 25. 147. The compound of claim 130, wherein M is, ateach occurrence, independently pyrene, perylene, perylene monoimide or6-FAM or derivative thereof.
 148. The compound of claim 130, wherein M,at each occurrence, independently has one of the following structures:


149. The compound of claim 130, having the following structure:

wherein: z is 3, 4, 5 or 6; m is 2, 3 4 or 5; n is an integer from 1 to10; and F″ has the following structure:


150. A composition comprising the compound of claim 130 and one or morebiomolecules.
 151. The composition of claim 150, wherein the biomoleculeis an enzyme, receptor, receptor ligand, antibody, bacteria, virus,blood cell or tissue cell.
 152. A method of staining a sample,comprising adding to the sample the compound of claim 130 in an amountsufficient to produce an optical response when said sample isilluminated at an appropriate wavelength.
 153. A method for visuallydetecting biomolecule, the method comprising: (a) admixing the compoundof claim 130 and the biomolecule, thereby associating the compound andthe analyte; and (b) detecting the compound by its visible properties.154. A fluorescent compound comprising Y number of fluorescent moietiesM, wherein the fluorescent compound has a peak fluorescence emissionupon excitation with a predetermined wavelength of ultraviolet light ofat least 85% of Y times greater than the peak fluorescence emission ofeach single M moiety upon excitation with the same wavelength ofultraviolet light, and wherein Y is an integer of 2 to 10, and whereinthe fluorescent compound is covalently bound to a targeting moiety at aterminal end of the fluorescent compound.
 155. The fluorescent compoundof claim 154, wherein each of the fluorescent moieties M independentlyhave one of the following structures:


156. The fluorescent compound of claim 154, wherein each M is covalentlybound to at least one other M by a linker comprising L¹, L², L³ and apolyethylene oxide, wherein the polyethylene oxide having the followingstructure:

L¹, L² and L³ are each independently alkylene or heteroalkylene; m is aninteger from 2 to 10; z is an integer from 2 to 30; and each

independently indicates a point of attachment to L¹, L² or L³.
 157. Thefluorescent compound of claim 154, wherein the targeting moietyselectively binds to a biomolecule.
 158. The fluorescent compound ofclaim 154, wherein the biomolecule is an enzyme, receptor, receptorligand, antibody, bacteria, virus, blood cell or tissue cell.
 159. Amethod for detecting a biomolecule, the method comprising: (a) admixingthe fluorescent compound of claim 154 and the biomolecule, therebyassociating the fluorescent compound and the biomolecule; and (b)detecting fluorescent emission from the fluorescent compound associatedwith the biomolecule.